Analysis and Purification of
Oligonucleotides by Anion Exchange and
Ion-Pairing Reversed Phase Chromatography
With an introduction by Valentina D’Atri, PhD
University of Geneva, Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO),
Geneva, Switzerland
Julia Bartmann, Dr. Daniel Eßer, Dr. Judith Wortmann,
YMC Europe GmbH

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography2Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

This Whitepaper …
…is written for all chromatographers who deal with theanalysis and / or purification of oligonucleotides. The
focus is on anion exchange chromatography (AEX) andion pair reversed phase chromatography (IP-RP).
For both modes, the main aspects are the selection ofa suitable stationary and mobile phase as well as the
optimum temperature.
Of course, the special features of the large-scale process
are also taken into account. If this aspect is of particularinterest to you, simply follow the icon:

In this whitepaper, you are guided through the following
aspects:

Introduction to Therapeutic Oligonucleotides
by Valentina D’Atri, PhD, University of Geneva
Separation Modes in Liquid Chromatography for Therapeutic Oligonucleotides
Anion Exchange Chromatography
Ion-Pairing Reversed Phase Chromatography
Important Considerations for Loadability and Scale-Up3

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

1. Introduction
By Valentina D’Atri, PhD (University of Geneva, Institute of Pharmaceutical Sciences of Western
Switzerland (ISPSO), Geneva, Switzerland)
Therapeutic oligonucleotides (TOs) are synthetic DNA or

be used to inhibit the activity of viral proteins or proteins

RNA oligomers designed to target specific genes or tran-that are overexpressed in cancer cells [6].

scriptional pathways with the purpose of treating a wide
range of diseases, including cancer, genetic disorders,and viral infections [1, 2, 3].

According to their size and complexity, TOs can be generally classified in short single-stranded oligonucleotides(ASOs, SSOs, gapmers, antagomirs, and agomirs), short

Under the name of TOs lies a wide range of differ-

double-stranded oligonucleotides (siRNA), and large sin-

ent nucleic acid structures including e.g. antisense

gle-stranded ONs (aptamers). Short oligonucleotides canoligonucleotides

oligo-

be produced by solid-phase synthesis, while enzymatic

nucleotides (SSOs), gapmers, antagomirs, and ag-

in vitro transcription (IVT) is preferred for larger oligonu-

omirs

cleotides [7].

(miRNA(ASOs)

asmimetics),

splice-switchingsmallinterfering

RNAs(siRNAs), and aptamers [4, 5].
The solid-phase synthesis of therapeutic oligonucleoThe different nucleic acid structures of TOs imply
different4functionsandmechanisms

oftides is a complex cyclic process that involves several

action:steps, including detritylation, coupling, and capping nu-i) ASOs are designed to bind to specific RNA sequences

cleotides of the growing oligomer, purification, and char-and inhibit the expression of disease-causing genes. They

acterisation. The oligonucleotides must be synthesised

can be used to silence the expression of oncogenes, viralwith high accuracy and purity, and they must be charac-

genes, or genetic mutations that cause inherited diseases.

terised to ensure that they are of the desired length andii) siRNAs are designed to bind to specific mRNA se-sequence [8]. Indeed, several impurities can be formed

quences and induce degradation of the mRNA, there-at each step of the synthesis, mainly consisting in short-byproteins,

mers (n–1, n–2), longmers (n+1), abasic oligonucleotides,

while iii) aptamers are designed to bind to specific pro-and partially deprotected or detritylated oligonucleotides

teins and alter their functions. Aptamers and siRNA can[8, 9, 10, 11, 12, 13].

inhibitingtheexpressionofspecificAnalysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

In addition, TOs can be chemically modified to enhance

A classification of common starting material andtheir stability, targeting ability, or other properties. Chemi-

process-related

cal modifications can be introduced at several sites of theotide

impurities

manufacturinghas

observedbeeninoligonucle-

recently

published

nucleotide, namely heterocyclic nucleobase, sugar moi-

by Rupp and Cramer with the goal to guide a chem-

ety, and phosphodiester linkage, as well as at the back-istry,

manufacturing,andcontrols

(CMC)

chem-

bone level (sugar and phosphodiester) [14, 15]. Some of

ist in oligonucleotide therapeutic development [20].

the most common chemical modifications include: phos-

Of note, most of the impurities require advanced analyti-

phorothioate (PS, replacing one of the oxygen atoms incal instrumentation to allow their identification and quan-

the phosphate backbone of the oligonucleotide with a sul-tification as they are closely related to the parent com-phur atom), which increases the stability and nuclease re-pound.

sistance of the oligonucleotide); sugar modification such

In this context, approaches based on liquid chromatog-

as 2'-O-Methyl (2'OMe), 2'-O-Methoxyethyl (2'MOE), andraphy (LC) have undoubtedly a pivotal role in the impu-

Locked Nucleic Acid (LNA), which increase the stability

rity characterisation and purification of oligonucleotides

and nuclease resistance of the oligonucleotide and also

and the advancement of CMC strategies to better monitor

increases its ability to bind to its target; and the phos-these impurities. Specifically, anion exchange (AEX) andphorodiamidate morpholino backbone (morpholino), a

ion-pairing reversed phase chromatography (IP-RP) tech-synthetic alternative to the natural nucleotides having anniques can be addressed as reference approaches.

aminoglycoside-like structure that increases nuclease resistance, stability and target binding ability [14, 15]. These
are just a few examples, but there are many other types ofchemical modifications that have been developed and arebeing researched for use in therapeutic oligonucleotides[14, 15].
In addition to chemical modifications, synthetic ligands
and carriers can be used for targeted delivery, with oligonucleotides conjugated to GalNAc or encapsulated inlipid nanoparticles (LNPs) being the most used delivery
approaches [16, 17, 18, 19].
Obviously, the different levels of chemical complexity imparted by the chemical modifications and the addition ofdelivery ligands/carriers result in a wider diversity of impurities and structural variability that needs to be addressed.
Concerning the regulatory aspects, TOs are consideredat the interface between small molecule drugs and biologics, as they are manufactured like new chemical entities but also share properties with biologics. For this reason, they do not benefit from clear guidelines, althoughsuggestions on the classification of impurities originatingfrom the manufacturing process can be found in the literature [8].

5Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

1.1. Separation Modes

Anion Exchange (AEX)
Chromatography
Separation Principle
The separation of oligonucleotides via anion exchange

phase. Mostly, eluents with high salt concentrations in thechromatography is based on the direct interaction of theelution buffer are employed. Alternatively, the pH of thenegative charged sugar-phosphate backbone with themobile phase can be changed. As a result, the interaction

positive charged modification of the AEX stationary phase.

dissociates and the oligonucleotide elutes from the sta-

This charge-charge interaction leads to the retention oftionary phase and is separated from other molecules withthe target molecule to the stationary phase. The elutiondifferent charge profiles.

Figure 1: Separation principle of AEX.

6

N
O

O

O

P

O
O

O

O

P

O

O

O

O

O

O

HN

HN

N

O

O

CH
3

CH
3

is initiated by increasing the ionic strength of the mobileAnalysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Ion-Pairing Reversed Phase (IP-RP)
Chromatography
Separation Principle
fective retention of the oligonucleotide on the RP phase.

able to interact with the stationary phase. Therefore ion-

The elution is performed by increasing the content of or-pair reagents with a hydrophobic group are required.

ganic modifier of the mobile phase (including the IP-rea-

They act as mediators between the charged target andgent), in order to elute the oligonucleotide-ion pair-com-

the hydrophobic stationary phase. This leads to an ef-

plex from the stationary phase.

N
O
O

O

O

O

O

P

O

O

P

O

O

O

O

O

HN

HN

N

O

O

CH
3

CH
3

Of course, the charged oligonucleotide backbone is not

H C
3

NH

CH
CH

3

H C
3
3

NH

CH

CH
3
3

Figure 2: Separation principle of IP-RP.

7

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

1.2. Analysis and Purification of Oligonucleotides
Analysis
The purpose of the analysis of oligonucleotides is the quantitative and / or qualitative characterisation of the target
molecules.

Aim: Obtaining Information About the Target Molecule or the Mixture
In analytical LC, the focus is on parameters that allow to

To obtain the required information from the chromatogram,

gain information as fast as possible from a small amountthe sensitivity of the system setup needs to be highof sample. Therefore, the resolution between the peaks ofbecause some components of the target mixture areinterest should be sufficient but does not need to be large.

only trace impurities that have to be detected. For highThe peak shape in analytical chromatography should be

reproducibility, the method and columns have to be very

as ideal as possible (asymmetry factors close to 1).

robust so that the results are reproducible and reliableindependent from possible experimental deviations.

Identification &Characterisation

Small column sizes

Analytical separation• Resolution• Peak shape• Sensitivity• Throughput• Robustness

Important factors

As analytical LC only deals with a small amount of sample,

As smaller particles in smaller column dimensions pro-the column dimensions are correspondingly small. The

vide higher efficiencies, the peaks obtained are sharper

inner diameters (IDs) used for analytical methods typical-

and therefore the resolution higher.

ly range from 1.0–4.6 mm ID. Columns are packed with

Mass spectrometry (MS) detection is often used as it

stationary phases with small particle sizes, sub 2 µm forprovides more information on the oligonucleotide sam-

UHPLC and 3 and 5 µm for HPLC. Decreasing the par-ple compared to UV. However, the MS-compatibility of

ticle size is an effective tool to improve the resolution.

the mobile phase has to be considered in order to obtaina reasonable sensitivity.

Summary of technical parameters for separations on analytical scale:length: 30–250 mm
small, typical dimensions:
1.0–4.6 mm
ID:				small, typically <2–5 µm✔ Particle sizes:
aliphatic modifications (IP-RP) or cationic functionalities (AEX)✔ Phase selectivity:
buffers/IP reagents for optimal sensitivity, MS-compatibility✔ Mobile phase:ambient or elevated temperatures✔ Temperature:
UV, MS✔ Detection:
low, depending on detection limit✔ Sample loading:✔ Column sizes:

8Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Purification
The purpose of purification processes using preparative chromatography is the isolation of a target substance from asample.

Aim: Collection of a Target Substance with Increased Purity
In preparative LC, the focus is on the isolation of the tar-In this context, the production efficiency is the determin-

get compound to reach a required purity. For the devel-ing factor in how much substance can be purified from theopment of industrial scale processes, a comprehensive

sample feed within a purification run. This depends on theprocess development is required to identify the most pro-purity level that has to be achieved.

ductive and cost-efficient method.

Isolation &Purification

Large column sizes

Preparative purification• Purity• Production
efficiency• Recovery• Safety• Costs

Important factors

The scale of purification processes can vary greatly.

phases is typically larger compared to analytical LC

This depends on the amount of target that has to be

in order to reduce the backpressure at larger scales.

purified. If for example only a few milligrams or grams of

The mobile phase needs to be safe, available in large

an oligonucleotide have to be purified, the setup of thequantities and preferably at low costs. The cost-efficiency

preparative system is small. The scale of the preparativeof preparative processes strongly relies on the mobile

column and the system increases with larger sample feedphase used and its consumption.

volumes.

For optimal productivity, the sample loading should be as

Column IDs up to 2 m can be used for preparative LC

high as possible with sufficient recovery levels to optimise

methods, depending on the actual need for the final target

the overall yield. The detection is only required for thecompound. The particle size of preparative stationary

collection of the target – not for its identification.

Summary of technical parameters for separations on preparative scale:✔ Column sizes:✔ Particle sizes:✔ Phase selectivity:✔ Mobile phase:✔ Temperature:✔ Detection:✔ Sample loading:larger, especially larger IDs depends on scalelarge, typically 7–50 µm
aliphatic modifications (IP-RP) or cationic functionalities (AEX)
large quantities, cost-efficiency and safety required
ambient, high temperatures possible depending on equipment
UV, no MS-compatible buffer systems required
as high as possible depending on resolution

Purification aspects within this whitepaper:This icon indicates important aspects and considerations for the purification of oligonucleotides within
the whitepaper.

9Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2. AEX
2.1. Stationary Phase
2.1.1. Functional Group
Quaternary amine groups, which are strong anion ex-

As they lose their anion exchange functionality at high pHchangers, are the gold standard of functional groups invalues, they are only suitable to a limited extend in oligo-

the analysis and purification of oligonucleotides. Strong

nucleotide chromatography.

anion exchangers are protonated over the entire pH
range allowing for the use of high pH values which areH

CH3

often applied in oligonucleotide separations. In contrast,

C

N

CH3

H

CH3

+

weak anion exchangers are not protonated over the entire
pH range.

Strong Anion Exchanger
Weak Anion Exchanger

2

3

4

5

6

7

8

9

10

11

12

pH

Figure 3: Protonation ranges of strong and weak anion exchangers.

2.1.2. Particle Technology
In the entire chain of oligonucleotide chromatography

10highly robust stationary phases. Polymer-based particles

extreme pH values and temperatures can be required.

provide high stability against both, high pH and temper-

Whilst during their analysis high temperatures are used toature which is why they are often preferred over silica

improve the resolution, the Cleaning-in-Place (CIP) afterparticles in oligonucleotide chromatography. Porous andpurification uses high pH values up to 14; both require

non-porous particles are commonly available.

Analytical scale(Semi) preparative scale

Non-porous polymer beads

Porous polymer beads

Non-porous particles are usually applied on an analyticalticles are the better choice. Thanks to the porous struc-scale due to their high efficiency at low loadings. Due toture the accessible surface area is increased allowing forthe enhanced mass transfer sharp peaks can be obtained.

a higher loadability. Figure 4 demonstrates the benefits at

For higher loadings required for purifications, porous par-different loading amounts.

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

BioPro IEX SF (non-porous type)

BioPro IEX SP (porous type)

AU
0.06

AU
0.06

High
y
ef ficienc

0.04
0.02

0.04
0.02

0.00

0.00

Column efficiency (Plates)

26

27

28

29

30

31 min

30

32

33

34

35 minColumn efficiency vs. Loading amount

70000

BioPro IEX SP
BioPro IEX SF

60000
50000
40000
30000
20000
10000
0

15

30

60

90

120

BioPro IEX SF
0.60

0.60

0.50

0.50

0.40

0.40

0.30

0.30

0.20

0.20

0.10

0.10
28

29

300

BioPro IEX SP
AU
0.70

27

150

Loading amount of Lysozyme (µg)

AU
0.70

0.00
26

31

30

31 minColumn: BioPro IEX SF/SP (5 µm) 50 x 4.6 mm ID
Part Nos.: SF00S05-0546WP / SPA0S05-0546WP
Eluent:A) 20 mM NaH2PO4-Na 2HPO4 (pH 6.8)
B) 20 mM NaH2PO4-Na 2HPO4 (pH 6.8) containing 0.5 M NaCl
Gradient: 0–100%B (0–60 min)

0.00
30

High
ty
loadabili
31

32

33

34

35 minFlow rate:
0.5 mL/min
Temperature: 25°C
Detection:
UV at 280 nm
Injection:
100 μL
Sample:Lysozyme

Figure 4: Column efficiency and loadability of non-porous and porous BioPro IEX columns.

Purification of Oligonucleotides: Particle Technology
For the efficient purification of oligonucleotides, IEX

capacities (DBC) and low backpressures that also en-

resins with optimised base materials are needed. The

able high resolution purifications are the key to suc-particles need to allow high sample loading and shouldcess. Hydrophilic polymer-based materials provide highhave good pressure-flow characteristics for high pro-chemical and mechanical stability and consequently are

ductivity. Therefore, resins with high dynamic binding

well-suited for oligonucleotide purification processes.

11Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2.1.3. Particle Size
When developing a new method, chromatographers havethe limitations of the equipment specification. In order toto consider the particle size. If there is a separation task

perform a smooth scale-up, it is very important to makefor analysis, small particle sizes such as 3 and 5 µm aresure that various particle sizes are available. If scouting

typically used. For purification purposes, larger particle

columns were packed with analytical particle size it issizes such as 10, 20 or 30 µm can be employed. It de-necessary to check that the corresponding preparative

pends on the final scale of the purification. The limiting

particle sizes provide the same selectivity to deliver con-factor is the resulting backpressure which must meetsistent resolution.

Purification of Oligonucleotides: Selecting the Most Suitable Particle Size
The purification of oligonucleotides requires very highFor large scale purifications, 30 µm particle sizes

resolution because highly similar impurities includ-

are suitable. Polishing steps and high-resolution

ing longmers and shortmers have to be removed. On

purifications are usually developed with 20 andthe other hand, the process has to be productive and10 µm particle sizes. State of the art resins suchcost-efficient. Therefore, resolution and reasonableasbackpressure need to be balanced.

retention profile and differ only in the resolution

IEX resins like YMC’s BioPro IEX SmartSep are availa-of the eluting peaks. As a result, the particle size canble in different particle sizes and allow full flexibility in

be changed during process development which allows

method development: 30 , 20 as well as 10 µm.

tailored solutions.

BioProIEX

SmartSep

providecomparable

BioPro IEX SmartSep Q10

BioPro IEX SmartSep Q20

BioPro IEX SmartSep Q30

0

5

10

15

20

25

30

35

Column Dimension: 50 x 4.6 mm ID
Resin Part Nos.:
BioPro IEX SmartSep Q10 (10 µm): QSA0S10
BioPro IEX SmartSep Q20 (20 µm): QSA0S20
BioPro IEX SmartSep Q30 (30 µm): QSA0S30
Eluent:A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 0.5 M NaCl
Figure 5: Scalability of BioPro IEX SmartSep.

12

40

45

50

55

min

Gradient:
0–80%B (0–60 min)Flow rate:
180 cm/h (0.5 mL/min)
Temperature: 30 °C
Detection:
UV at 220 nm
Sample:Transferrin and trypsin inhibitor

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Practical Example: Purification of Oligonucleotides with Different Particle Sizes
Figure 6 shows the separation of a DNA 20mer oligo-

opment because different particle sizes can be tested.

nucleotide using two different particle sizes (10 µm andIf the particle sizes are scalable, which means they pro-

30 µm). The resolution of both traces differs significant-vide the same retention behaviour, a change of the par-ly, whereas the retention profile of both separations re-ticle size can easily be implemented without modifying

mains the same. Resins that are available in different

the separation parameters.

particle sizes allow more flexibility in process devel-

UV intensity– BioPro IEX SmartSep Q10– BioPro IEX SmartSep Q30

0

5

10

15

20

25

30

35

Retention time [min]Column Dimension: 100 × 4.6 mm ID
Resin Part Nos.:
BioPro IEX SmartSep Q10 (10 µm): QSA0S10
BioPro IEX SmartSep Q30 (30 µm): QSA0S30
Eluent:A) 10 mM NaOH
B) 10 mM NaOH + 1 M NaClO4
Gradient:
25–40%B (0–30 min), 40–100%B (30–35 min)Flow rate:
1 mL/minTemperature:
25 °C
Detection:
UV at 260 nm
Injection:
5 µL
Sample:Trityl-OFF 5‘-ATA CCG ATT AAG CGA AGT TT-3‘ (DNA 20mer)
Figure 6: Purification of 20mer DNA oligonucleotide using 10 and 30 µm BioPro IEX SmartSep.

13

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Important Considerations for Process Scale Up
The smaller the particle size of the stationary phase, theto an elevated backpressure at higher linear flow rates.

higher is the resulting backpressure. This correlation

With adequate hardware and system, small particles areshould be considered at an early stage of process de-well-suited for very high-resolution purifications. This re-velopment. The systems and column hardware used forquires systems and column hardware with higher pres-the purification of oligonucleotides determine the pres-sure ratings that can cope with the higher backpressure

sure limit. Figure 7 illustrates the pressure-flow curves ofof the small particles. If low pressure equipment is usedthe different particle sizes of the BioPro IEX SmartSep– especially for very large-scale applications – larger par-resins. As shown in the diagram, the 10 µm particles leadticles are the preferred option.

5 cm x 0.5 cm ID

Backpressure [MPa]3– BioPro IEX SmartSep 10 µm– BioPro IEX SmartSep 20 µm– BioPro IEX SmartSep 30 µm

2.5
2.0
1.5
1.0
0.5
0

0

200

400

600

800

Linear ﬂow rate [cm/h]Figure 7: Pressure-flow characteristics for BioPro IEX SmartSep.

Important Check List
Before choosing a phase for a method screening in preparative LC,
the following check list might be helpful:

1. Optimal particle technology
2. Variable particle sizes available
3. Reproducibility
4. Scalability
5. Supply guarantee

14

1000

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2.2. Mobile Phase
2.2.1. Choice of Buffer and Counter Ion
In addition to the stationary phase, the mobile phase has

increased from 8.1 up to 12 which was beneficial fora great influence on oligonucleotide chromatography.

the ionisation states of the oligonucleotide, resulting in

During method development, it is worth trying different

improved tailing factors.

buffers and salt types as both can significantly influence

In a second step, the counter ion was changed in orderpeak shape and the subsequent resolution. Different

to improve the tailing factor further. By replacing NaClbuffers and salt combinations are suitable, but Tris-HCl

with NaClO4 the tailing was further suppressed and higher

and NaOH based buffers mixed with NaCl and NaClO4

sensitivities could be obtained. The gradient profile forare typically used.

NaClO4 was adjusted because the elution strength of

Figure 8 shows the comparison of Tris-HCl and NaOH

NaClO4 is two to three times greater than that of NaCl inbuffer. The sample used is an RNA 20mer sample. By

IEX. Especially, when high resolution is required the choiceselecting the NaOH buffer, the mobile phase pH wasof an appropriate buffer and salt can be very beneficial.

2 Salt type1 Buffer type
X=20 mM Tris-HCl (pH 8.1)
tR: 7.14
W0.5h: 0.090
TF: 1.95

mAU

30

20

10

10

0

0
10

20

30 min

X=10 mM NaOHtR: 9.20
W0.5h: 0.111
TF: 1.60mAU

30

tR: 9.20
W0.5h: 0.111
TF: 1.60

30

20

0

Y=2.0 M NaCl

mAU

0

30

20

10

10

0

20

30 minY=2.0 M NaClO4
tR: 11.14
W0.5h: 0.068
TF: 1.36

mAU

20

10

0
0

10

20

30 minEluent:A) X
B) X containing 2.0 M NaCl
Gradient: 15–100%B (0–30 min)

0

10

20

30 minEluent:A) 10 mM NaOH
B) 10 mM NaOH containing 2.0 M Y
Gradient: 15–100%B (0–30 min) for NaCl
5–50%B (0-30 min) for NaClO4

Column:BioPro IEX QF (5 µm) 100 x 4.6 mm ID
Part No.:QF00S05-1046WP
Flow rate:
1.0 mL/min
Temperatur: 25 °C
Detection: UV at 260 nm
Injection:
2 µL (10 nmol/mL)
Sample:5’-UCAUCACACUGAAUACCAAU-3’ (RNA 20mer)
Figure 8: Improved peak shapes by using different buffer and salt types.

!

Check the pH stability of your sample if you want to work with high pH mobile
phases to prevent degradation, especially for long oligonucleotides.

15

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2.2.2. Ionic Strength
The previous example already emphasised the impor-ry-over effects. However, it must be noted that a high salt

tance of using of an appropriate salt type. In addition, theconcentration can decrease the solubility of the analyte

ionic strength plays a key role. It controls secondary in-

and decrease the retention time.

teractions. Higher initial ionic strength can prevent car-––		

DNA 20mer Analytical chromatogram
DNA 20mer Blank chromatogram 			with Milli-Q injection after analysis

mAU

mAU

Carry-over

No Carry-overA) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
5–70%B (0–15 min), 74%B (15–18 min), 5%B (18–33 min)A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
40–70%B (0–15 min), 74%B (15–18 min), 40%B (18–33 min)Initial : 50 mM NaCl

Initial : 400 mM NaCl

Column:Part No.:
Flow rate:
Temperature:
Detection:
Injection:
Sample:

BioPro IEX QF (5 μm) 100 x 4.6 mm ID
QF00S05-1046WP
1.0 mL/min
25°C
UV at 260 nm
2 µL (10 nmol/mL)
5‘-TCATCACACTGAATACCAAT-3‘ (DNA 20mer)

Figure 9: Influence of initial ionic strength on carry-over behaviour.

Purification of Oligonucleotides:
PS Modified Oligonucleotides Require High pH and Ionic Strength16

A challenging task is the purification of phosphoro-tion. In contrast, the same conditions lead to a massive

thioate (PS) oligonucleotides. Their elevated negative

increase in retention for the PS modified oligonucleo-

charge leads to greater retention on the AEX resin.

tide. These conditions are not suitable for purification

The following example shows the elution profile of anof the molecule. Therefore, the pH as well as the ionic

DNA oligonucleotide with a complete phosphodiester

strength of the mobile phase needed to be increased.

backbone (PO) and a phosphorothioate backbone. The

The

modifiedconditions

with

10 mM

NaOH

PO oligonucleotide shows good elution results using

and 2 M NaClO4 gave to an improvement of thea slightly alkaline buffer (pH 8.0) and 1 M NaCl for elu-

oligonucleotide elution.

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

pH 8
1 M NaCl

pH 8
1 M NaCl

PS

UV intensity

UV intensity

PO

0

5

10

15

20

25

30

0

35

5

10

Retention time [min]15

20

Retention time [min]25

30

increased pH andelution strength

35

PS

UV intensity

pH 11
2 M NaClO4

0

5

10

15

20

25

30

35

Retention time [min]Column Dimension: 100 × 4.6 mm ID
Resin Part No.:
BioPro IEX SmartSep Q10 (10 µm): QSA0S10
Eluent:A) 10 mM Tris (pH 8)
B) 10 mM Tris (pH 8) containing 1 M NaCl or 10 mM NaOH containing 2 M NaClO4
Gradient:
20–70%B (0–30 min), 70–100%B (30–35 min) or
35–70%B (0–30 min), 70–100&B (30–35 min) or
20–40%B (0–30 min), 40–100%B (30–35 min)Flow rate:
0.2 mL/min
Temperature:
25 °C
Detection:
UV at 260 nm
Sample:5‘-TCT CTA TCA ATA TCG TGG-3‘ (DNA 18mer)
Figure 10: Different requirements for the elution of PO and PS oligonucleotides.

For the purification of PS modified oligonucleotides, high pH and ionic strength are beneficial and can improve theelution of such high-binding molecules.

!

The total net charge increases with the oligonucleotide length as more negatively
charged phosphates are added to the oligonucleotide backbone. This is why especially long oligonucleotides require a high ionic strength at the end of the gradient.

2.2.3. Organic Modifier and Temperature
Elevated temperature and the use of an organic modifier

fer and minimises the influence of the oligonucleotides’

can influence the oligonucleotide chromatography (Figure

secondary structure on the retention.

11). Phosphorothioated RNA samples with only a single

In the following steps, methanol was added to the mobile

base difference in length are ideally suited to show thephase and the gradient was adjusted.

benefit. By increasing the temperature from 25 to 60 °C

Both the resolution and sensitivity could be improved andnarrower peaks and improved resolution can be obtained.

even narrow peaks with partly resolved variants could be

The elevated temperature enhances the mass trans-

detected.

17

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

mAU

15

10

2
1

5

0
0

5

10

15

min

mAU

from
25°C
to
60°C

15

10

1

2

5

0
0

5

10

mAU

1

15

min

2

additionofmethanol

15

10

5

0
0

5

10

Column:Part No.:
Eluent:15

BioPro IEX QF (5 μm) 100 x 4.6 mm ID
QF00S05-1046WP
Upper and middle chromatogram:A) 10 mM NaOH/methanol (100/0)
B) 10 mM NaOH containing 1.0 M NaClO4/methanol (100/0)
Bottom chromatogram:A) 10 mM NaOH/methanol (70/30)
B) 10 mM NaOH containing 1.0 M NaClO4/methanol (70/30)
Gradient:
Upper and middle chromatogram:
32–80%B in 8 min
Bottom chromatogram:
40–100%B in 6.3 min
Flow rate:
1.0 mL/min
Temperature: Upper and middle chromatogram:
25 °C
Bottom chromatogram:
60 °C
Detection:
UV at 260 nm
Injection:
2 μL (10 nmol/mL)
Sample:1: RNA 20mer 5’-U~C~A~U~C~A~C~A~C~U~G~A~A~U~A~C~C~A~A~U-3’
2: RNA 21mer 5’-G~U~C~A~U~C~A~C~A~C~U~G~A~A~U~A~C~C~A~A~U-3’~phosphorothioate

Figure 11: Influence of temperature increase and the addition of organic modifier.

18

min

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Purification of Oligonucleotides: Elevated Temperatures
Elevated temperatures can also be beneficial for pre-gonucleotide itself and the benefit of elevated temper-

parative processes. If the overall system allows elevat-

atures has to be considered carefully regarding theed temperatures, the elution of the oligonucleotide canoverall cost-efficiency and the thermal stability of thebe improved, especially for strongly binding molecules.

target oligonucleotide.

However, the optimal temperature depends on the oli-

Purification of Oligonucleotides: Organic Modifiers for Improved Elution
Generally, aqueous buffer systems are used for the pu-

purities. Typically, acetonitrile or isopropanol is add-rification of biomolecules via IEX but for the purifica-

ed and their benefit on the elution is investigated. Thetion of oligonucleotides, high demands on the separa-

process environment, the preparative system as welltion efficiency are required. The addition of an organicas the column hardware and the resin itself have to bemodifier to the mobile phase can improve the elution

compatible with such conditions.

depending on the oligonucleotide and the critical im-

2.2.4. Gradient Slope
Every oligonucleotide is different and so are the require-

The trade-off between sharp peaks and high resolution

ments for the chromatographic separation. This is why

has therefore to be considered thoroughly, especiallythe method development is more complex compared towhen thinking of oligonucleotide purifications. High res-the development of separation methods for other target

olution means higher efficiency and any increase in reso-

molecules. In principle, fast and steep gradients provide

lution is beneficial. Using a shallower gradient, the reso-

ever, the peaks become broader and elution takes longer.

base and in length (20 and 21mer).

Ionic strength

lution might be increased by 19% (see Figure 13) for DNA
samples with differences in the type of base of 5’ terminal

Ionic strength

sharp peaks but the peaks are eluted closely together.
By decreasing the slope, the resolution increases, how-

Elution volume

Elution volume

Figure 12: Influence of gradient slope on resolution and peak shape.

19

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

25–55%B (0–15 min)
Δ=2%B/min

G*

mAU

2

1

50

25–40%B (0–15 min)
Δ=1%B/min

RS: 6.90

40

/

30
20
10
0
0

A*

5

mAU

10

1

3

50

15 minmAU

RS: 2.46

40

40

30

30

20

20

10

10

0
5

10

15 min

mAU

0

5

10

15 min

mAU

14

50

RS: 1.70

RS: 2.02

50

40

40

30

30

20

20

10

10

0

1 4

0
0

T*

1 3

0
0

C*

RS: 2.85

50

5

mAU

10

1

50

5

15 min0

5

10

15 minmAU

RS: 3.73

RS: 4.40

50

40

40

30

30

20

20

10

10

0

1

5

0
0

5

10

15 min0

5

10

15 minColumn:Part No.:
Eluent:

BioPro IEX QF (5 μm) 100 x 4.6 mm ID
QF00S05-1046WPA) 10 mM NaOH
B) 10 mM NaOH containing 1.0 M NaClO4
Flow rate:
1.0 mL/min
Temperature: 25 °C
Detection:
UV at 260 nm
Injection:
4 μL (5 nmol/mL each)
Sample:1: 5’-TCATCACACTGAATACCAAT-3’ (DNA 20mer)
2: 5’-GTCATCACACTGAATACCAAT-3’ (DNA 21mer)
3: 5’-ATCATCACACTGAATACCAAT-3’ (DNA 21mer)
4: 5’-CTCATCACACTGAATACCAAT-3’ (DNA 21mer)
5: 5’-TTCATCACACTGAATACCAAT-3’ (DNA 21mer)*base of 5’end of DNA 21mer

Figure 13: Gradient optimisation for DNA samples with 5' terminal base differences.

Purification of Oligonucleotides: Gradient Optimisation
Detailed information on the optimisation of gradient elution in IEX can be found in the whitepaper about
method development using IEX.

20

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2.3. Cleaning-in-Place
Efficient Cleaning-in-Place (CIP) procedures are needed

itise the column. Typically, high alkaline solutions up tofor productive and cost-efficient oligonucleotide purifica-

1 M NaOH are used. These conditions are highly effective

tions. The purification of biomolecules such as oligonucle-

to increase the column lifetime. Consequently, process

otides leads to non-specific adsorption and precipitation

resins that are stable towards the continuous applicationon the resin over time. Aqueous native buffers add theof these conditions are needed.

risk of microbial growth. Cleaning procedures with alka-

The regularly applied flushing with alkaline solutions hasline solutions remove the precipitations and non-specifican additional benefit: endotoxins, a major impurity in oli-

adsorbed substances efficiently and additionally san-gonucleotide purification are efficiently removed.

CIP Study

Determination of Dynamic Binding Capacity

Cleaning-in-Place procedure (Repeated 10 times)
a)
b)
c)
d)
e)

1.0 M NaCl aq. (5 CV)
1.0 M NaOH aq. (5 CV, Contact time: 1 h)
1.0 M NaCl aq. (2 CV)
H2O (2 CV)
Equilibration buffer of DBC measurement (5 CV)

DBC (mg/mL-resin)

120
100
80
60

BioPro IEX SmartSep Q30

40
20
0

0

10

20

30

40

50

60

70

80

90

100

Number of CIP cycle

Column Dimension: 50 × 5.0 mm ID
Resin Part No.:		
BioPro IEX SmartSep Q30 (30 µm): QSA0S30
Equilibration buffer: 20 mM Tris-HCl (pH 8.6)
Elution buffer:		
Equilibration buffer containing 0.5 M NaCl

Flow rate:
200 cm/h (0.66 mL/min)
Temperature: 25 °C
Detection:
UV at 280 nm
Sample:
1.5 mg/mL BSA in equilibration buffer

CIP cycle
Column Dimension: 50 × 5.0 mm ID
Flow rates:
200 cm/h (1.0 M NaCl, H2O, Buffer)
30 cm/h (1.0 M NaOH)
Temperature:
25 °C
Figure 14: Stability of BioPro IEX SmartSep Q30 under alkaline CIP conditions.

21

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

2.4. YMC Stationary Phases for AEX
BioPro IEX
QF

BioPro IEX
QA

Ion Exchange Type

non-porous
3; 5porous5

10
Cl –pH Range

2.0–12.0

Temperature Range

4–60 °C

2030

3.0 MPa

3.0 MPa

3 µm: 18–25 MPa
5 µm: 6–12 MPa

2.5–3.5 MPa

pre-packed
columnspre-packed
columns

bulk resin + pre-packed
columns

Ion Exchange
Capacity

N/A

N/A

min. 0.08 meq/mL-resin

Dynamic Binding
Capacity

N/A

N/A> 100 mg BSA/mL Resin

0.4–0.5 mL/min(144–181 cm/h)*

200–1,000 cm/hMax. 2,000 cm/h

Available Formats

Typical Flow Rate

* for a 100 x 4.6 mm ID column

22

quaternary amine

Counter Ion

Pressure Limit

BioPro IEXSmartSep Q30

strong anion exchanger

Charged Group

Particle Size [µm]BioPro IEXSmartSep Q20

hydrophilic polymer(polymethacrylate)

Base Particle

Pore Size [nm]BioPro IEXSmartSep Q10

5 µm:
0.2–0.8 mL/min(72–289 cm/h)*
3 µm:
0.2–0.5 mL/min(72–181 cm/h)*

4.0 MPaAnalysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3. IP-RP
3.1. Stationary Phase
3.1.1. Particle Technology
For IP-RP oligonucleotide separations, stationary phases

the preferred choice, due to their higher mechanical andwith different base particles can be used. Fully porous

chemical stability. High pH mobile phase conditions oror core-shell based silica and hybrid silica particles are

performing alkaline cleaning studies oblige to use hybrid

the most typical phases used. Hybrid silica particles are

silica resins.

1

2

3

4

5

6

7

8

9

10

11

12

pH

Silica pH Range
Hybrid Particle pH Range
Figure 15: Applicable pH range for silica and hybrid silica particles.

3.1.2. Modifications and Selectivity
The selectivity is the most powerful tool to increase theAlso, different C18 columns provide varying results

resolution. In IP-RP oligonucleotide separations, alkyl

as shown for all PO RNAs in Figure 16. Pore size andphases are most commonly used, especially C18 or C8hydrophobicity play an important role, which is further

modified phases but also C4 phases may be required

discussed in section 3.1.3.

sometimes.

All PO RNA

Resolution

mAU
15

YMC-Triart C18 ExRS (8 nm pore)

I.S.

10

Rs (1,2) = 9.8
Rs (2,3) = 7.8
Rs (3,4) = 1.6

4

5

3

2

1

0
0

1

2

3

4

5

6

7

8

9

min

mAU

Rs (1,2) = 12.4
Rs (2,3) = 9.9
Rs (3,4) = 2.2

15

YMC-Triart C18 (12 nm pore)

10

4

I.S.

3

2

1

5
0
0

1

2

3

4

5

6

7

8

9

min

mAU

Rs (1,2) = 11.3

15

I.S.

YMC-Triart Bio C18 (30 nm pore)Rs (2,3) = 10.9
Rs (3,4) = 2.7

4

10

1

5

3

2

0
0

1

2

Columns: YMC-Triart C18 ExRS (3 µm, 8 nm) 50 x 2.1 mm ID
YMC-Triart C18 (3 µm, 12 nm) 50 x 2.1 mm ID
YMC-Triart Bio C18 (3 µm, 30 nm) 50 x 2.1 mm ID
Part Nos.: TAR08S03-05Q1PTH
TA12S03-05Q1PTH
TA30S03-05Q1PTH
Eluent:A) 8 mM TEA, 200 mM HFIPB) methanol
Gradient: 10–20%B (0–10 min)

3

4

5

6

7

8

9

min

Flow rate:
0.42 mL/min
Temperature: 65°C
Detection:
UV at 260 nm
Injection:
1 µL
Sample:1. 5’-CACUGAAUACCAAU-3’ (14mer)
2. 5’-UCACACUGAAUACCAAU-3’ (17mer)
3. 5’-UCAUCACACUGAAUACCAAU-3’ (20mer)
4. 5’-GUCAUCACACUGAAUACCAAU-3’ (21mer)
I.S. caffeine

Figure 16: Influence of C18 modification of all PO RNA analysis.

23

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Even though C18 columns are most commonly used,

When using the C18 column, the target could not be elut-

considering other selectivities can be helpful when fac-

ed as the hydrophobic character of the disulfide units

ing challenging analytes. In Figure 17 the separation ofrequire a less hydrophobic stationary phase. Good peakdisulfide modified oligonucleotides is shown using both ashapes could be obtained using a widepore C4 phase.

polar C18 and a C4 stationary phase.

AU

YMC-Triart Bio C4
Target

＊

0.08
0.06
0.04
0.02
0.00
0

5

10

15

20

25

30

35

40

min

40

min

AU
0.20

polar C18 phase

0.15

＊
0.10

Target is not eluted.

0.05

0.00
0

5

10

Column:5 µm, 250 x 4.6 mm ID
Product code: TB30S05-2546PTH
HS12S05-2546WT
Eluent:A) 50 mM TEAA* (pH 7.0)/acetonitrile (95/5)
B) acetonitrile
Gradient:
5–95%B (0-30 min), 95%B (30–35 min),
95–5%B (35–35.1 min), 5%B (35.1–45 min)Flow rate:
1 mL/min

15

20

25

30

35

Temperature: 50 °C
Detection:
UV at 260 nm
Sample:Crude reaction mixture*Triethylammonium acetate
By courtesy of Saki Kawaguchi, Chemistry Department,
Nagoya University, Japan

Figure 17: Separation of oligonucleotides modified with disulfides using C18 and C4 modified stationary phases.

24

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.1.3. Pore Size
The stationary phase pore size has a big influence on theneed to be evaluated. In general, the pore size is recom-

chromatographic results in RP processes, as it deter-

mended to be as the smallest possible and the largest

mines the accessibility of the specific surface area andnecessary. Generally, for short single-stranded oligonu-

influences the hydrophobicity of the stationary phase. On

cleotides without any bulky modifications, a smaller pore

the one hand, the pore has to be large enough so that thesize is recommended. For larger oligonucleotides up to

target oligonucleotide can enter the pores. On the othermultiple hundreds of nucleotides in length, larger pore

hand, it needs to be small enough in order to allow sharp

sizes are required for an efficient separation. The porepeaks and a good resolution.

size selection tool (Figure 18) gives a general overview on

As oligonucleotides can vary in their length from just a few

the recommended pore size based on the length of sin-

up to multiple hundred nucleotides, different pore sizes

gle-stranded oligonucleotides.

Length (mer)

12 nm pore

20 nm pore

30 nm pore

> 300
100
50
40
30
20
most appropriate MW range

10

appropriate MW range

Figure 18: Pore size selection tool.

These values are only recommendations and the limits are only approximate, as potential structures or modifications
can lead to different requirements. Therefore, the testing of different pore sizes can be useful.

25Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

In this example, several pore sizes have been tested for

non-accessibility due to the small pore size. The 12 nm

10–120mer poly dT DNA strands. It showed that the 8 nm

pore size showed comparable behaviour to 30 nm for shortpore was inappropriate for oligonucleotide separation atoligonucleotides. For 30mer or more, the 30 nm pore wasall, as it showed an increased peak half width for the en-

the best choice as the oligonucleotides could fully perme-tire oligonucleotide lengths. This may be caused by theate the pore and interact with the bonded C18 phase.

I.S. (caffeine)
30

60

25
20

10

40

15

Pore size
100

80

0.16

120

120

(YMC-Triart C18 ExRS)

12 nm(YMC-Triart C18)

Peak half width (min)

8 nm

0.14

100

10mer

0.12

25

15

0.10

20

12 nm

60

40

30

0.08

30 nm

30 nm
(YMC-Triart Bio C18)
0

5

10

15

20

25

min

8 nm

80

0.06

0

10,00020,00030,00040,000

MW Oligonucleotide (Da)Columns:

YMC-Triart C18 ExRS (3 µm, 8 nm) 50 x 2.1 mm ID
YMC-Triart C18 (3 µm, 12 nm) 50 x 2.1 mm ID
YMC-Triart Bio C18 (3 µm, 30 nm) 50 x 2.1 mm ID
Part Nos.:
TAR08S03-05Q1PTH
TA12S03-05Q1PTH
TA30S03-05Q1PTH
Eluent:A) 4 mM TEA, 100 mM HFIPB) methanol
Gradient:
5–20%B (0–30 min)Flow rate:
0.42 mL/min
Temperature: 65 °C
Detection:
UV at 260 nm
Injection:
1 µL (0.2–2.0 µM)
Sample:Poly dT (10–120mer)
Figure 19: Influence of the stationary phase pore size on peak width.

Remember that the molecular weight of double-stranded oligonucleotides can be!

nearly double the size of a single-stranded oligonucleotide with the same length.
Modifications increase the molecular weight further. Potential secondary structuresof the molecule can have an additional influence on the pore accessibility.

26

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.1.4. Particle Size
It is well known that by decreasing the particle size, the resolution increases, but with an increase in the backpressure.
In analytical scale, 3 and 5 µm particle sizes are typically used for HPLC, whereas sub 2 µm particles are used for highly
sensitive UHPLC analyses.

Purification of Oligonucleotides: Selection of the Appropriate Particle Size
In preparative chromatography, particle sizes such as

the preparative system that is used for purification.

7, 10 or 20 µm are commonly used in RP separations.

A stationary phase available with different particle siz-Particle sizes such as 7 or 10 µm provide higher res-es provides an increased flexibility in IP-RP processes

olution, but with higher backpressures (see chapter

development. YMC-Triart (Prep) for example is availa-

2.1.3.). Therefore, the selection of the optimal particle

ble with completely scalable particle sizes – from ana-

size depends on the required resolution as well as onlytical to preparative scale.

Important Check List
Before choosing a stationary phase for analytical methods or
purification processes, the following check list might be helpful:

1. Optimal particle technology
2. Variable particle sizes available
3. Reproducibility
4. Scalability
5. Supply guarantee

27Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.2. Mobile Phase
3.2.1. Choice of Buffer System
Sensitivity and resolution strongly depend on the mobiled(pT)2–20 using different buffer systems. HFIP-TEA pro-phase composition. Therefore, not every buffer system isvides much higher sensitivity and better resolution com-appropriate for use in oligonucleotide chromatography.

pared to triethylammonium acetate (TEAA). However, it isOn an analytical scale, the use of fluoroalcohols with alkyl

immiscible with acetonitrile.

amines such as HFIP-TEA (1,1,1,3,3,3-hexafluoro-2-pro-

Alternative alkyl amine acetates such as DBAA (dibu-

panol-triethylamine) is the gold standard as it provides

tylammonium acetate) or TEAA buffers can also be used,

a good sensitivity and reproducible chromatographic

but higher buffer concentrations may provide increased

results in combination with UV detection as well as withresolution and stronger retention (see Figure 21). On the

MS detection. Figure 20 shows the UV (red) and MS (or-contrary, lower concentrations reduce system contamina-ange) traces of separations of oligodeoxythymidylic acidtion in (U)HPLC-MS analyses.

10 mM TEAA (pH 6.0)

400 mM HFIP-TEA (pH 7.0)

UV detection(at 269 nm)

22–25% methanol (0–20 min)15

100 100

50 50

50

0 0
25 min 0 0

20

MS detection(ESI Negative)

15

100100

20

20

20

28

10 10

15 15

20 20

50

50

0
0
25 min 0
25 min
0

5

5

10

10

15

15

20

20

0
25 min25 min 0

100 100

100

50 50

50

50

50

0

0

0 0
25 min 0 0

5

5

10 10

15 15

20 20

Hydrosphere C18 (3 µm, 12 nm) 50 x 2.0 mm ID
HS12S03-0502WT
0.2 mL/min
UV at 269 nm (red), ESI negative mode (orange)
35°C
5 µL (25 pmol/mL)
Oligo(deoxythymidylic acid) [d(pT)2–20]

25 min 0
25 min25 min 0

0

5

5

10 10

15 15

20 20

0

5

5

10

10

15

15

20

20

0

25 min 0
25 min

100

100

50

50

Figure 20: Signal
intensity in UV and MS detection using different buffer systems.
0
0 0

!
15

5

100

100100Column:Part. No.:100100
Flow rate:
Detection:
Temperature:
50 50
Injection
Sample:1557–35% methanol (0–20 min)25 min 0
25 min

5

10

15

20

25 min0100100

100

100

50 50

50

50

In order to prevent contamination and to ensure reproducible results, buffers

5

5

0

5

0

5

should be prepared daily.
0 0
25 min 0 0

5

5

10 10

15 15

20 20

0
25 min 0
25 min

5

10

15

20

25 min0Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Purification of Oligonucleotides:
Cost-Efficient Buffer Systems and IP Reagents
100

100

50

50

For preparative applications, economic buffer systems are required, such as DBAA or TEAA. Generally, the
0

0

5

10

15

20

0

25 min05

cost-efficiency and safety are the major factors that influence the selection of suitable buffer systems and
100

100

50

50

IP-reagents.
methanol
22–25% methanol (0–20 min)15

20

10 mM DBAA (pH 6.0)

10 mM TEAA (pH 6.0)

100

15

20

0

0

20

10
15
15

15
20
20

10

15

20

0

25 min15
20

10–13% acetonitrile (0–20 min)

0

0

0

25 min

100

50

5
0

10
5

15
10

20
15

0

25 min20

0

0

25 min

50

50

50

50

5

0
0

0
0
5
5
100

0
0

0
0
0

10
15
15

15
20
20

20
25 min25 min0 0
025 min

0

00
5
0
5
100
100

5
10
10

10
15
15

15
20
20

20
25 min
min
25

0
0 0
0

25 min

0
0

0
5
0
5

0

0

25 min00
00

25 min

100

15
20
15
20

20
25 min20
25 min

5
10
5
10

10
15
10
15

15
20
15
20

20
25
20 min25 minHydrosphere C18 (3 µm, 12 nm) 50 x 2.0 mm ID
HS12S03-0502WT
0.2 mL/min
UV at 269 nm (red), ESI negative mode (orange)

0

0

0 0
0

0

0
5

5
10

10
15

15
20

0
55

25 min25 min

50
50

50
50

25 min25 min

0
0

0
05
5

20
25 min25 min

0

50

50

0
5

5
10

10
15

15
20

50

0

0
0

0

25 min

5
10
10

10
15
15

15
20
20

20
25min
min
25

00
00

0

0
5

5
10
10

0
55

5
10
10

5
100

100
100

50

25 min

0

100
100

5
10
5
10

10
15
10
15

15
20
15
20

20
25 min20
25 min25 min25 min

0
0 0
0

5
5

100
100

5
10
5
10

10
15
10
15

15
20
15
20

20
25 min20
25 min25 min25 min

0
0

0
0

5
5

50

50

0

100
100

20
25 min25 min

10
5

50
50

100

25 min

15
20
20

5
0

100

15–37.5% acetonitrile (0–20 min)

0
0 0
5
0
5

100
100

100

0

10
15
15

0

50
50

100

0

25 min

50
50

100
100

0
0 0
0

5
10
10

10
5

100

50

25 min

0

0

50
50

0
0
5
5
100

5
0

Temperature: 35°C
Injection
5 µL (25 pmol/mL)
Sample:Oligo(deoxythymidylic acid) [d(pT)2-20]

100

0

0

25
20min

50
50

50

10
15
10
15

20
15

15–37.5% acetonitrile (0–20 min)

100
100

50

5
10
5
10

15
10

50

100

50
50

50
50

0

50
50

50
50

0

0

10
5

50

100
100

33.6–56% methanol (0–20 min)

100
100

50

5
10
10

5
0

100

50
50

100
100

50

20min
25
25 min

0
5
5

0

0

100
100

50

50

25 min0

25 min

50

50
50

20min
25

20

33.6–56% methanol (0–20 min)

100
100

0
0

15

100

50
50

25 min

10

5

50

50

0

0

100

100

100
100

20min
25

20

50

Figure 21: Signal intensity in UV and MS detection using different mobile phases.

10
15

15

100

0

20min
25

10

100

100

15
20

acetonitrile

100

Column:Part. No.:
Flow rate:
Detection:
10
15

5

100

25 min5 mM DBAA (pH 6.0)

15
20

5

0

100

50

100

15

100

50

100

10
15

0

20
25 min25 min

0

0

5

10

15

20

25 min

0

5

10

15

20

25 min29

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Purification of Oligonucleotides:
Efficient Desalting to Remove Buffer Salts Using RP
The purification of oligonucleotides requires the use of

A big advantage can be a stationary phase which re-(buffer-) salts and IP reagents. If these salts need to betains the target molecule under preferably 100% aque-removed from the final product, the use of a RP col-ous conditions such as YMC-Triart Prep C18-S and

umn is a convenient strategy. Using the same column

Phenyl-S. This will allow a cost-efficient desalting pro-for both the separation and desalting saves investment

cess as post-treatment after the purification run to becosts and lowers the footprint of the process.

applied. The general principle is shown in Figure 22.

Fraction

Adsorption/Desalting
Load

Desorption

Wash

RP column

TargetSalt
Figure 22: General desalting principle using an RP column.

Desalting / Resalting
The detailed procedure of a desalting process using RP can be found in the technical note:
Desalting/Resalting using a RP column.

30Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.2.2. High pH Stable Resins
In addition to the buffer composition and the selection of

The example below shows a screening of various concen-

the most appropriate ion-pair reagent, the pH of the mo-

trations of TEA and HFIP for all PO and PS RNA (Figure

bile phase is an important factor to influence the elution

24). In a first step, the concentration of HFIP was heldof the target compound. Depending on the target oligo-

constant at 100 mM, whilst the concentration of TEA wasnucleotide, mobile phases with high pH can improve thevaried between 1 and 30 mM resulting in pH values be-

elution. However, alkaline conditions are not possible fortween 7.4 and 8.9. Between 1 and 8 mM TEA, retention

conventional silica-based RP stationary phases. There-

increased for both all PO and PS RNA with increasing pH.

fore, hybrid silica-based RP phases with an extended pH

Using higher concentrations of 15 or 30 mM TEA, reten-

range are a great tool for efficient oligonucleotide separa-tion decreased again and resolution decreased which is ations – for analysis and preparative purifications.

commonly observed for PO/PS oligonucleotides.

All PO RNAAll PS RNA
1,2

1-4

I.S.

1 mM TEA-100 mM HFIP
pH 7.4W0.5(2) = n. c.
Rs(3,4) = n. c.
1-4

Rs(1,2) = n. c.
12

I.S.

2 mM TEA-100 mM HFIP
pH 7.7

W0.5(2) = n. c.
Rs(3,4) = n. c.

Rs(1,2) = 0.88

4
1

I.S.

4 mM TEA-100 mM HFIP
pH 8.01, I.S.

4

1

Rs(1,2) = 1.07
1 2

8 mM TEA-100 mM HFIP
pH 8.33

2

W0.5(2) = 0.0329
Rs(3,4) = 2.99

1 2

3

2

W0.5 (2) = 0.0328
Rs(3,4) = 2.81

W0.5(2) = 0.0314
Rs(3,4) = 2.01

W0.5: half width (min)Rs: resolution (JP)n. c.: not calculated
I.S.: internal standard

Rs(1,2) = 1.06

4

I.S.

1 2

15 mM TEA-100 mM HFIP
pH 8.63

2

1,2

Rs(1,2) = 0.92

1-4

30 mM TEA-100 mM HFIP
pH 8.9I.S.

W0.5(2) = n. c.

Rs(3,4) = n. c.
0Column:Part No.:
Eluent:

0.5

1

1.5

2

2.5

min

YMC-Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PT
A) bufferB) methanol
Gradient:
Initial: 8%B, gradient slope: 1%B/min
Flow rate:
0.42 mL/min
Temperature: 65 °C
Detection:
UV at 260 nm
Injection:
1 µL

Rs(1,2) = 0.43
0

1

2

3

4

5 min

Sample:all PO RNA:
1. 5’-CACUGAAUACCAAU-3’ (14mer), 1.8 nmol/mL
2. 5’-UCACACUGAAUACCAAU-3’ (17mer), 1.8 nmol/mL
3. 5’-UCAUCACACUGAAUACCAAU-3’ (20mer), 1.8 nmol/mL
4. 5’-GUCAUCACACUGAAUACCAAU-3’ (21mer), 3.6 nmol/mL
I.S. = caffeine
all PS RNA:
1. 5’-U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (20mer), 1.0 nmol/mL
2. 5’-G*U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (21mer), 1.0 nmol/mL*=PhosphorothioatedFigure 23: Influence of the IP concentration on All-PO and All-PS RNA separations.

!

As TEA has a pKa value of 10.7, the number of ionised TEA and therefore theion pairing functionality decreases at higher pH. Therefore, the optimal pH for
analysing oligonucleotides using a TEA-HFIP system is pH=8.

31

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

In a second step, separations with varying TEA and HFIP

RNA, peak shape got better and baseline resolution wasconcentrations at a pH around 8 were performed. This

achieved with 8 mM TEA – 200 mM HFIP or higher con-showed that higher TEA-HFIP concentrations provide

centrations. Please note that the results are based on UV

better resolution. Whilst high TEA concentrations improvedetection. Lower concentrations may be more favourable

the retention, high HFIP concentrations are required tofor MS detection due to ion suppression.

keep the pH value as low as possible. Especially for all PS

All PO RNA
mAU

mAU
6

I.S.

30

Rs(3,4) = 3.56

4

20

0

0

30

Rs(3,4) = 3.27

2

4

4

I.S.
1

20

15 mM TEA-400 mM HFIP
(pH 7.8)

3

2

1

10

mAU

All PS RNA

6

8 mM TEA-200 mM HFIP
(pH 7.9)

3

2

0
0
mAU
30

Rs(3,4) = 2.99

20

2

I.S
1

4

6

3

8 mM TEA-100 mM HFIP
(pH 8.3)

10

mAU

0
0

30

Rs(3,4) = 2.81

20

2

I.S.
1 2 3

4

6

0
0

4 mM TEA-100 mM HFIP
(pH 8.0)

30
20

2

4

2 mM TEA-50 mM HFIP
(pH 8.0)
4

6

4

6

8

1 2

Rs(1,2) = 1.82
0

2

4

6

8

YMC-Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PT
A) bufferB) methanol
Gradient:
Initial: 8%B, gradient slope: 1%B/min
Flow rate:
0.42 mL/min
Temperature: 65 °C
Detection:
UV at 260 nm
Injection:
1 µL

Rs(1,2) = 1.06

2
0

0

2

4

6

8

12

4

Rs(1,2) = 1.07

2
0

2

4

6

8

1,2

4

Rs(1,2) = n. c.

Peak splitting

2
0

2

4

6

8

min

Sample:all PO RNA:
1. 5’-CACUGAAUACCAAU-3’ (14mer), 1.8 nmol/mL
2. 5’-UCACACUGAAUACCAAU-3’ (17mer), 1.8 nmol/mL
3. 5’-UCAUCACACUGAAUACCAAU-3’ (20mer), 1.8 nmol/mL
4. 5’-GUCAUCACACUGAAUACCAAU-3’ (21mer), 3.6 nmol/mL
I.S. = caffeine
all PS RNA:
1. 5’-U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (20mer), 1.0 nmol/mL
2. 5’-G*U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (21mer), 1.0 nmol/mL*=PhosphorothioatedFigure 24: Influence of different TEA-HFIP concentrations.

12

4

0

min

Column:Part No.:
Eluent:

32

0

mAU
6

I.S.

2

2

2

0

6

1-4

10
0
0

0

4

mAU
6

10

mAU

Rs(3,4) = n. c.

4

0

mAU
6

4
2

Rs(1,2) = 2.39

2

mAU
6

10

1 2

4

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.3. Temperature
The use of elevated temperature is a common tool to

However, separation was improved at higher temperature.

improve peak shape and resolution. Increased temper-

For all PS RNA, elevated temperatures were essential toature influences the secondary structure and provides

obtain sharp peaks and good resolution. At 45°C, accept-

enhanced dispersion and distribution velocity of an oli-able results were obtained, and 65°C were the best con-gonucleotide.

ditions. At higher temperatures, such as 90°C peak dete-

Figure 25 shows the separations of all PO and PS RNA

rioration was observed, which probably occurred due to ausing 25°C, 45°C, 65°C and 90°C. For all PO RNA, goodstructural change of the RNA sample.

separation is achieved even at lower temperature.

All PO RNAAll PS RNA

I.S.

mAU

4

30

Rs(3,4) = 3.80

20

initial 4%B

10
0

mAU
6

1

2

90ºC

2

4

65ºC
0

2

4

6

8

4

Rs(1,2) = 2.39

2initial 8%B

6

0

2

4

6

8

mAU
6

45ºC

10

4

Rs(1,2) = 1.64

2initial 12%B

0
0

2

4

6

mAU
6

mAU
30

0

2

4

6

8

4

Rs(3,4) = 3.02 20

25ºC

10
0

4

0

Rs(3,4) = 3.31 20

initial 15%B

2

mAU
6

10

0

initial 4%B
0

mAU
30

initial 12%B

Rs(1,2) = 3.33

2

6

Rs(3,4) = 3.56 20
0

1

0
0

mAU
30

initial 8%B

2

4

3

Rs(1,2) = 1.16

2

initial 16%B

0
0

2

4

6

min

Column:Part No.:
Eluent:YMC-Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PT
A) bufferB) methanol
Gradient:
Initial: 8%B, gradient slope: 1%B/min
Flow rate:
0.42 mL/min
Temperature: 65 °C
Detection:
UV at 260 nm
Injection:
1 µL

0

2

4

6

8

min

Sample:all PO RNA:
1. 5’-CACUGAAUACCAAU-3’ (14mer), 1.8 nmol/mL
2. 5’-UCACACUGAAUACCAAU-3’ (17mer), 1.8 nmol/mL
3. 5’-UCAUCACACUGAAUACCAAU-3’ (20mer), 1.8 nmol/mL
4. 5’-GUCAUCACACUGAAUACCAAU-3’ (21mer), 3.6 nmol/mL
I.S. = caffeine
all PS RNA:
1. 5’-U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (20mer), 1.0 nmol/mL
2. 5’-G*U*C*A*U*C*A*C*A*C*U*G*A*A*U*A*C*C*A*A*U-3’ (21mer), 1.0 nmol/mL*=PhosphorothioatedFigure 25: Temperature influence on All-PO and All-PS RNA separations.

Purification of Oligonucleotides: High Temperature Purifications
The benefit of increased temperatures on the sepa-quantities have to be heated so that the benefit ofration efficiencies of oligonucleotides becomes morehigher temperatures on the overall productivity andimportant for preparative applications. Elevated tem-the economics of the process have to be considered.

peratures can improve the separation efficiency andIn the end, the cost-efficiency is the determining factor

therefore the productivity of oligonucleotide purifica-

and this depends on the overall purification processtions but require special equipment. For purificationand the value of the target substance.

processes larger column volumes and mobile phase33

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.4. Flow Rate
The adaption of the flow rate is a powerful tool to adjust

improves the productivity because more substance canthe throughput of a method. At analytical scale, a higher

be purified in the same time. The maximum flow rate isflow rate decreases the cycle time and consequently al-

generally limited by the resulting resolution and backpres-lows more analytical runs per day because analysis timesure. Therefore, the optimal flow rate of a method has tois shortened. At preparative scale, the higher throughput

be evaluated.

Purification Aspects: Higher Flow Rates for Higher Productivity
The following example shows two purification runs of thesure increases from 0.8 MPa (8 bar) to 1.7 MPa (17 bar).

same sample at two different flow rates. The collected

Although the yield in case of higher flow rate was low-

fractions with the required purity of ≥98% are marked

er, the overall productivity could be significantly in-in yellow. With the elevated flow rate, the backpres-creased as shown in the table below.

Fraction with ≥ 98% purity
0.8 l/min
Pressure = 0.8 MPa

0

10

20

30

40

50

60

70

80

Fraction with ≥98% purity
1.6 l/min
Pressure = 1.7 MPa

0

10

20

30

Time (min)40
50Time (min)0.8 mL/min

Flow Rate

1.6 mL/min

60 minCycle time

30 min

1 run

Run/Hour2 runs30.1 mg/g gel

Loading30.1 mg/g gel

71.8%

Theoretical Yield

64.7%

17.1 mg/g gel

Productivity/Hour

30.8 mg/g gel

60

70

80

Figure 26: Higher flow rates influence the productivity.

This example illustrates the influence of the flow rate on the overall productivity. To identify the optimal flow rate, ascreening of different flow rate conditions is required.

34

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.5. Detection
The liquid chromatography of oligonucleotides on analyti-trol. Determining the oligonucleotide structure is required

cal scale is typically coupled to mass spectrometry due toover the entire process to ensure the patient’s safety. In

its benefits over other detection techniques. MS provides

contrast to AEX, RP is easier to couple with MS due to thehigh accuracy and sensitivity for high throughput anal-

absence of non-volatile salts in high amounts. In additionyses. LC-MS analyses are applied over the entire chain,

to MS, UV detection is often used which is also the state

during the oligonucleotide development up to quality con-of the art detection for preparative chromatography.

A) UV

mAU
20

21mer

22mer

10
0

B) MS

0.0
Int.(x100000)
5

2.5

5.0

7.5

10.0

12.5

15.0

17.5

min

MIC
(m/z 1721.5, m/z 1798.1)
21mer

22mer

0
0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

min

Courtesy of M. Yamada, SHIMADZU CORPORATION

5’-pUGG AGU GUG ACA AUG GUG UUG-3’
5’-pUGG AGU GUG ACA AUG GUG UUG U-3’

(21mer, MW 6,890.1)(22mer, MW 7,196.3)

Column:Part No.:
Eluent:YMC-Triart C18 (3 µm, 12 nm) 150 x 2.0 mm ID
TA12S03-1502WTA) 10 mM di-n-butylamine-acetic acid (pH 7.5)
B) 10 mM di-n-butylamine-acetic acid (pH 7.5)/acetonitrile (50/50)
Gradient:
62–72%B (0–20 min)Flow rate:
0.2 mL/min
Temperature: 30 ºC
Detection:A) UV at 260 nm
B) ESI-negative mode
Injection:
4 µL (5 nmol/mL)
System:LC) Shimadzu ProminenceMS) Shimadzu LCMS2020
Figure 27: UV and MS detection of miRNA analysis.

35

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.6. Bioinert Column Hardware
Due to the negatively charged phosphate groups in the

The most common columns for metal coordination ana-

backbone of the oligonucleotides, they are able to inter-lytes either have a bioinert coating or a PEEK-lining inside

act with positively charged metal ions of the conventional-

the stainless steel body such as YMC-Accura Triart andly used stainless steel column hardware. The coordination

YMC-Triart metal-free, respectively. Both column types

with metals can lead to a poor peak shape and decreased

provide an increased sensitivity and high recovery with-recovery. Both can be improved by the use of a special

out any preconditioning necessary.

column hardware which minimises these interactions.

!

Optimum results are achieved by using both a bioinert column and system. However,as the column accounts for 70% of the metallic surface of the flow path its change
has the biggest influence on the chromatographic results.

YMC-Accura Triart Bio C18

mAU

6

1

6

2

d
Increasetyi
sensitiv h
and hig !
recovery42Standard column

mAU

4

2

0

0

7

8

min

7

Column:Part No.:
Eluent:YMC-Accura Triart Bio C18 (1.9 µm, 30 nm) 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 15 mM triethylamine - 400 mM HFIP*B) methanol
Gradient:
8–18%B (0–10 min)Flow rate:
0.42 mL/min
Temperature: 65º C
Detection:
UV at 260 nm
Injection:
1 µL
Sample:All PS RNA 20mer (1) (5’-U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’)
All PS RNA 21mer (2) (5’-G^U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’)^=Phosphorothioate*1,1,1,3,3,3-hexafluoro-2-propanol

Figure 28: Increased sensitivity and high recovery using bioinert column hardware.

36

2

1

8

min

mAU
1000Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

500
3.7. Cleaning-in-Place
Cleaning-in-Place procedures can massively improve thecolumn with alkaline solutions (e.g. 0.1 M NaOH) restorescolumn lifetimes in preparative RP-processes. Problems

the column performance. This is only possible with sta-and solutions are similar as for the purification
0 of biomol-

0.0of the1.0
ecules via IEX (see chapter 2.3.). The washingRPtionary phases that provide a high pH stability.

2.0

3.0

4.0

5.0

6.0 min

YMC-Triart Prep Bio200 C8(10 µm, 20 nm)

mAU
1000
mAU
1000
500

Initial

500
0
0

CIP

After
300
cycles

0.0

1.0

2.0

3.0

4.0

5.0

6.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0 min

mAU
1000
mAU
1000

G o od
peak
shape

500
500
0
0

Figure 29: Cleaning-in-Place stability after 300 cycles.

0.0

1.0

2.0

3.0

4.0

5.0

6.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0 min

Purification of Oligonucleotides: YMC Triart-Prep

mAU
1000
Further information on CIP-procedure with pH stable RP stationary phases can be found in the
Application Note about the CIP-Stability of YMC-Triart Prep.

500

0

0.0

1.0

2.0

3.0

4.0

5.0

6.0 min37

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

3.8. YMC Stationary Phases for IP-RP
Analysis of Oligonucleotides: Stationary Phases for Analytical Separations
YMC-Triart
C18
Base Material
Modification

YMC-Triart
Bio C18

inorganic/
inorganic/organic hybrid silica organic hybrid silica

Hydrosphere
C18
silicaYMC-Triart
C8

YMC-Triart
Bio C4

inorganic/
inorganic/organic hybrid silica organic hybrid silica

C18 (USP L1)

C18 (USP L1)

C18 (USP L1)

C8 (USP L7)

C4 (USP L26)

1.9, 3, 5

1.9, 3, 5

2, 3, 5

1.9, 3, 5

1.9, 3, 5

Pore Size [nm]

12

30

12

12

30

Spec. Surface Area [m2 /g]

360

–

330

360

–

1.0–12.0

1.0–12.0

2.0–8.0

1.0–12.0

1.0–10.0

Temperature Range

pH < 7: 90°C
pH > 7: 50°C

pH < 9: 90°C
pH > 9: 50°C

50 °C

pH < 7: 90°C
pH > 7: 50°C

pH < 7: 90°C
pH > 7: 50°C

Available Formats*pre-packed
columns:
SST, bioinert
coated SST or
PEEK-lined SST

pre-packed
columns:
SST, bioinert
coated SST or
PEEK-lined SST

pre-packed
columns:
SST

pre-packed
columns:
SST, bioinert
coated SST or
PEEK-lined SST

pre-packed
columns:
SST, bioinert
coated SST or
PEEK-lined SST

Particle Size [µm]

pH Range*SST: stainless steel

Purification of Oligonucleotides: Bulk Stationary Phases for Preparative Scale
YMC-Triart Prep
C18-S

YMC-Triart Prep
C8-S

Base Material
Modification

YMC-Triart Prep
C4-S

YMC-Triart Prep
Phenyl-S

inorganic / organic hybrid silica
C18

C8

C8

C4

Phenyl7, 10, 15, 20

10, 15, 20

10

10

10

Pore Size [nm]

12

12

20

12

12

Spec. Surface Area [m2 /g]

360

360

proprietary

360

360

Particle Size [µm]

pH Range
Available Formats

38

YMC-Triart Prep
Bio200 C8

2.0–10
CIP: pH 12.0
bulk resin + pre-packed columns

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

4. Loadability and Scale-up
4.1. Loadability Studies at Analytical Scale
After the successful method development at small scale,maximum amount of sample that can be purified in eachloadability studies at analytical scale are the next step

cycle. The loadability can be increased in different ways

towards a productive oligonucleotide purification. Dur-to achieve an overloading effect that leads to efficient re-ing this step, the sample load is increased to identify thecovery results.

Analytical
Load

Preparative
Load

After fractionating:purity control

Purity99.2%

Figure 30: Purity control after performing analytical loading and loadability studies.

4.2. Scale-Up to Preparative Scale
If the method is completely developed including the iden-

ticle size of the stationary phase, the separation modetification of the optimal loading conditions, the final setup

and the column length, remain the same. To increase thecan easily be transferred to the final process scale. This

final scale, the diameter of the preparative column is in-is done by applying the linear scale up. Within these pro-creased and the flow rate is adjusted according to thecedures, all non-scalable parameters, including the par-scaling factor (Figure 31).

Purification of Oligonucleotides: Linear Scale-Up
A detailed guideline can be found in the Technical Note about linear scale-up in preparative LC method
development.

Factor (SF)SF =AID prep.

ID2prep.

=
AID analyt. ID2analyt.

Column length determines retention time
Diameter determines loadability
Figure 31: Scale-Up Factor calculation.

39

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

4.2.1. Practical Example:
Scale-Up of an Oligonucleotide Purification ProcessThis practical example shows the purification of a syn-

20 mm and the column length remained the same. To

thetic 30mer oligonucleotide using a C18 modified sta-follow the rules of the linear scale-up, the flow rate wastionary phase. The analytical run is performed using aadjusted to 19 mL/min according to the scaling factor of50  x  4.6 mm ID column and the resulting chromatogramapprox. 19. The loading was increased at the same scale.

shows the main peak and the impurity peaks. The flow

The resulting chromatogram is completely comparable torate at this scale was set to 1.0 mL/min. For the prepara-

the analytical one: the peak shape and the retention time

tive purification, the ID of the column was increased toof the target peak are nearly the same.

Analysis 1.0 mL/min, 5 µL injection
Hydrosphere C18
50 x 4.6 mm ID, 5 µm

Purification 19 mL/min, 100 µL injection
YMC-Actus Hydrosphere C18
50 x 20 mm ID, 5 µm
Recovery 56%

30mer

Impurities

purity>99%
Part Nos.:

HS12S05-0546WT
HS12S05-0520WX
Eluent:A) 10 mM DBA-acetic acid (pH 6.0) / methanol (60/40)
B) 10 mM DBA-acetic acid (pH 6.0) / methanol (20/80)
Gradient:
10–35%B (0–30 min.)Temperature: ambient
Detection:
UV at 269 nm
Sample:synthetic oligonucleotide (100 µM)
Figure 32: Easy purification with YMC-Actus semi prep columns.

40

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

5. Conclusion
Therapeutic oligonucleotides are synthetic DNA or RNA

Even though the goals of oligonucleotide analyses andoligomers that can be quite diverse, not only in their func-

purifications are quite different, it is important to consid-

tion but also in their structure. Different separation modes

er them together in order to design an efficient process.

can be applied for oligonucleotide LC, but the current gold

Whereas small column dimensions and particle sizes are

standards these days are anion exchange and ion-pairingused to identify and characterise oligonucleotides in an-reversed phase chromatography. Additional modes suchalytical LC, large column IDs and particles are used foras HILIC (hydrophilic interaction chromatography) or SEC

their isolation and purification. This leads to different im-(size exclusion chromatography) are also gaining more in-portant factors which play a role:terest as alternative approaches.

Analytical LC

Preparative LC

Resolution
Peak shapeSensitivity
Sample throughput
Method robustness

PurityProduction efficiency
Recovery
SafetyProcess costs

Similar to the different aspects to be considered in analytical and preparative scale, selected parameters havea significant influence in AEX and IP-RP mode:

AEX

IP-RP

Functional Group

Strong or weak exchanger

C18, C8, C4, Phenyl

Particle Technology

Porous or non-porous polymer(Hybrid) silica

Particle Size

3-5 µm (analytical)
10-30 µm (preparative)<2 – 5 µm (analytical)
7–50 µm (preparative)

Mobile Phase

Tris-HCl or NaOH +NaCl or NaClO4
MeOH as organic modifier

HFIP-TEA (analytical)
DBAA or TEAA (analytical, preparative)

Temperature

25–60°C

25–90°C

HardwareColumn Cleaning

Bioinert or stainless steel
CIP using NaOH

Despite the different aims in analytical and preparative LC

ing study can be performed in analytical scale followedof oligonucleotides, often analytical methods can be scaled

by a scale-up to the final preparative dimensions andup for preparative purposes or are the starting point for amethod parameters. YMC offers robust AEX and IP-RP

preparative application. This scale-up is a straight-forward

stationary phases to analyse and purify oligonucleotides.

approach as long as a linear scale-up is applicable, there-

YMC is your preferred supplier addressing all required

fore the same selectivity is provided by a stationary phase

chromatographic parameters for analytical and prepara-

independent from its particle size. As a result, the load-

tive chromatography of oligonucleotides!

41

Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

Literature[1] Harries, L. W. RNA Biology Provides New Therapeutic Targets for Human Disease. Front. Genet. 2019,		10. https://doi.org/10.3389/fgene.2019.00205.
[2] Rinaldi, A. RNA to the Rescue. EMBO Rep. 2020, 21 (7). https://doi.org/10.15252/embr.202051013.
[3] Dammes, N.; Peer, D. Paving the Road for RNA Therapeutics. Trends Pharmacol. Sci. 2020,		41 (10), 755–775. https://doi.org/10.1016/j.tips.2020.08.004.
[4] Vanhinsbergh, C. J. Analytical Separation Methods for Therapeutic Oligonucleotides. LC/GC Eur. 2020, 33 (10), 20–26.
[5] Hammond, S. M.; Aartsma-Rus, A.; Alves, S.; Borgos, S. E.; Buijsen, R. A. M.; Collin, R. W. J.; Covello, G.; Denti, M. A.; Desviat,
		L. R.; Echevarría, L.; Foged, C.; Gaina, G.; Garanto, A.; Goyenvalle, A. T.; Guzowska, M.; Holodnuka, I.; Jones, D. R.; Krause, S.;
		Lehto, T.; Montolio, M.; Van Roon-Mom, W.; Arechavala-Gomeza, V. Delivery of Oligonucleotide-based Therapeutics:
		Challenges and Opportunities. EMBO Mol. Med. 2021, 13 (4). https://doi.org/10.15252/emmm.202013243.
[6] Smith, C. I. E.; Zain, R. Therapeutic Oligonucleotides: State of the Art. Annu. Rev. Pharmacol. Toxicol. 2019, 59 (1), 605–630.
		https://doi.org/10.1146/annurev-pharmtox-010818-021050.
[7] Kwon, H.; Kim, M.; Seo, Y.; Moon, Y. S.; Lee, H. J.; Lee, K.; Lee, H. Emergence of Synthetic MRNA: In Vitro Synthesis
		of MRNA and Its Applications in Regenerative Medicine. Biomaterials 2018, 156, 172–193. https://doi.org/
		10.1016/j.biomaterials.2017.11.034.
[8] Capaldi, D.; Teasdale, A.; Henry, S.; Akhtar, N.; den Besten, C.; Gao-Sheridan, S.; Kretschmer, M.; Sharpe, N.; Andrews, B.;
		Burm, B.; Foy, J. Impurities in Oligonucleotide Drug Substances and Drug Products. Nucleic Acid Ther. 2017, 27 (6), 309–322.
		https://doi.org/10.1089/nat.2017.0691.
[9] Chen, D.; Yan, Z.; Cole, D. L.; Srivatsa, G. S. Analysis of Internal (n-1)mer Deletion Sequences in Synthetic Oligodeoxyribo		nucleotides by Hybridization to an Immobilized Probe Array. Nucleic Acids Res. 1999, 27 (2), 389–395. https://doi.org/10.1093/
		nar/27.2.389.
[10] Fearon, K. L.; Stults, J. T.; Bergot, B. J.; Christensen, L. M.; Raible, A. M. Investigation of the ‘n–1’ Impurity in Phosphorothioate
		Oligodeoxynucleotides Synthesized by the Solid-Phase β-Cyanoethyl Phosphoramidite Method Using Stepwise Sulfurization.
		Nucleic Acids Res. 1995, 23 (14), 2754–2761. https://doi.org/10.1093/nar/23.14.2754.
[11] Temsamani, J.; Kubert, M.; Agrawal, S. Sequence Identity of the n-1 Product of a Synthetic Oligonucleotide.
		Nucleic Acids Res. 1995, 23 (11), 1841–1844. https://doi.org/10.1093/nar/23.11.1841.
[12] Krotz, A. H.; Klopchin, P. G.; Walker, K. L.; Srivatsa, G. S.; Cole, D. L.; Ravikumar, V. T. On the Formation of Longmers in 				Phosphorothioate Oligodeoxyribonucleotide Synthesis. Tetrahedron Lett. 1997, 38 (22), 3875–3878. https://doi.org/10.1016/		
		S0040-4039(97)00798-3.
[13] Septak, M. Kinetic Studies on Depurination and Detritylation of CPG-Bound Intermediates during Oligonucleotide Synthesis.
		Nucleic Acids Res. 1996, 24 (15), 3053–3058. https://doi.org/10.1093/nar/24.15.3053.
[14] Eckstein, F. Phosphorothioates, Essential Components of Therapeutic Oligonucleotides. Nucleic Acid Ther. 2014, 24 (6), 		
		374–387. https://doi.org/10.1089/nat.2014.0506.
[15] Iwamoto, N.; Butler, D. C. D.; Svrzikapa, N.; Mohapatra, S.; Zlatev, I.; Sah, D. W. Y.; Meena; Standley, S. M.; Lu, G.;
		Apponi, L. H.; Frank-Kamenetsky, M.; Zhang, J. J.; Vargeese, C.; Verdine, G. L. Control of Phosphorothioate Stereochemistry
		Substantially Increases the Efficacy of Antisense Oligonucleotides. Nat. Biotechnol. 2017, 35 (9),		845–851. https://doi.org/10.1038/nbt.3948.
[16] Kaczmarek, J. C.; Kowalski, P. S.; Anderson, D. G. Advances in the Delivery of RNA Therapeutics: From Concept to Clinical 				Reality. Genome Med. 2017, 9 (1), 60. https://doi.org/10.1186/s13073-017-0450-0.
[17] Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F. RNA-Targeted Therapeutics. Cell Metab. 2018, 27 (4), 714–739. https://		doi.org/10.1016/j.cmet.2018.03.004.
[18] Springer, A. D.; Dowdy, S. F. GalNAc-SiRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics.
		Nucleic Acid Ther. 2018, 28 (3), 109–118. https://doi.org/10.1089/nat.2018.0736.
[19] Foster, D. J.; Brown, C. R.; Shaikh, S.; Trapp, C.; Schlegel, M. K.; Qian, K.; Sehgal, A.; Rajeev, K. G.; Jadhav, V.; Manoharan, M.;
		Kuchimanchi, S.; Maier, M. A.; Milstein, S. Advanced SiRNA Designs Further Improve In Vivo Performance of GalNAc-SiRNA
		Conjugates. Mol. Ther. 2018, 26 (3), 708–717. https://doi.org/10.1016/j.ymthe.2017.12.021.
[20] Chapter 9 - CMC and regulatory aspects of oligonucleotide therapeutics, Rupp and Cramer, RNA Therapeutics, 2022,
		pages 263-320, https://doi.org/10.1016/B978-0-12-821595-1.00012-9.

42Analysis and Purification of Oligonucleotides byAnion Exchange and Ion-Pairing Reversed Phase Chromatography

FURTHER INFORMATIONYMC BROCHURESThe Selectivity Company

The Selectivity Company

YMC
Biochromatography
Columns

Reliable · Robust · Reproducible

Hybrid silica-based

BioProIEX Resins( U)HPLC columns
YMC-Triart

stationary phases for prep LC

YMC-Triart Prep

RP
SEC
IEX
HIC

Efficiency
Reliability
pH Stability

LC/MS(U)HPLC
SFC

Antibodies
Oligonucleotides
Proteins/Peptides

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Columns

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BioPro IEX Resins

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Prep

TECH NOTESTechnical Note

Technical Note

Technical Note

HILIC analysis of oligonucleotides using different
bioinert columns

How to choose the optimum conditions for analysis
of denatured or non-denatured siRNA duplexes

How to influence oligonucleotide analysis by IP-RP(AEX) seem to be the gold standards for the characterisationof oligonucleotides and their by-products. In addition,hydrophilic interaction liquid chromatography (HILIC) can
provide an alternative approach due to the highly polar natureof oligonucleotides.

Oligonucleotides have become more important for medical
applications, currently they are used to treat several diseases.
Therefore, robust and highly sensitive analysing methods
are required. Meanwhile ion pair reversed phase liquid
chromatography (IP-RP) and anion exchange chromatography

conditions so that both sense and antisense strands aremonitored. Analysis of siRNA under denaturing and nondenaturing conditions can be achieved by ion pair reversed
phase high performance liquid chromatography (IP-RP
HPLC). Several parameters influence siRNA separation:

Small interfering RNAs (siRNAs) usually occur as
oligonucleotide duplexes. They can be analysed under nondenaturing conditions so that duplex formation is assured
and excessive single-strands are detected. Alternatively,
analysis of siRNAs can be conducted under denaturing

1. Column temperature – key factor for the denaturation of siRNA
2. Ion pair reagents and acids – affect double-strand stability and separation efficiency

Expert tip
The phosphate-rich backbone of siRNA molecules enables the siRNA to adhere to metal surfaces, includingcolumn hardware. To prevent metal surface interaction, all experiments were conducted with a bioinert
YMC-Accura Triart Bio C18 column. The bioinert surface coating of column body and frit considerablyincreases the sensitivity and sample recovery.

Technical NoteThe natural structure of siRNA with an electron-richanionic phosphate backbone hampers its cellular uptake and is responsible for a poor pharmacokinetic profile.
The additional resistance towards nuclease-mediateddegradation led to the development of chemical modifications of the nucleotides structure. A common option is theintroduction of synthetic artificial linkages such as phosphorothioate linkages (PS) instead of the natural phosphodiester linkages (PO). The exchange of the oxygen
atom with sulphur in PS linkages decreases the polarityof the nucleotides and sensitivity to enzymatic degradation. As a result, PS oligonucleotides are more commonly
synthesized and used for in vivo and in vitro applications.
The chemical synthesis of oligonucleotides is a multistep
process, where the nucleotide chain is extended succes-

sively. During synthesis, impurities with only minor structural differences such as shortmers (n-x) and longmers(n+x) can accumulate. Therefore, the purity and qualityof oligonucleotides needs to be controlled closely since
impurities can affect their efficacy. The analysis of oligonucleotides is highly challenging due to their negatively
charged phosphate groups in the backbone. Ion pair reversed phase liquid chromatography (IP-RP) is considered
the gold standard for oligonucleotide analysis. The selection of several parameters such as pH, gradient and column temperature highly influences the performance. Also
the choice of ion pair reagent, pore size and hardware
plays an important role. This technical note demonstrates
the most important factors that need to be consideredregarding the analysis of oligonucleotides by IP-RP.

This technical note introduces all relevant aspects of denaturing and non-denaturing IP-RP HPLC. As a sample thesiRNA duplex targeting the firefly luciferase gene GL2 was used (table 1). The siRNA consists of 19 nucleotides.

O

Table 1: Oligonucleotide sequencesiRNA5’-CGU ACG CGG AAU ACU UCG AdTdT-3’
3’-dTdTGCA UGC GCC UUA UGA AGC U-5’

O-

Bioinert column options
Their main difference is their hydrophobicity: YMC-Accura
Triart columns are less hydrophobic compared to YMC-Triart
metal-free columns. Further, YMC-Accura Triart columns offer
a more robust end fitting without any preferences regarding
connection – in contrast to the PEEK-lined columns which
require special connecting options such as MarvelXACT
connectors. These are also available with a bioinert PEEK
layer or a PEEKsil capillary.

A much more robust and simple solution is to use a fullybioinert system and bioinert column hardware. The column
body represents more than 70% of the surface that theanalytes get in touch with [1]. Consequently, a bioinert
column body and frits will provide a distinct improvement in
performance. Therefore, YMC offer two bioinert hardware
options: YMC-Accura Triart (U)HPLC columns with a bioinert
coating of the column body and frits and YMC-Triart metalfree (U)HPLC columns with a PEEK-lining and PEEK frits.

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email: info@ymc.de · www.ymc.eu

Table 2: Overall chromatographic conditions unless otherwise stated.

Column:Part No.:
Eluent:Gradient:
Flow rate:
Temperature:
Detection:
Injection:
Sample:XO
In figure 1, the different states of the siRNA at different temperatures are visible. At an elevated temperature of 65 °C,
the double-stranded oligonucleotide is separated into twosingle-strands. The retention corresponds to the two single-strands analysed separately. At 45 °C the siRNA is partially degraded and the retention time increases. At 25 °C
non-denatured siRNA is detected, which elutes significantly
later from the stationary phase.

At high temperatures, double-stranded oligonucleotides
dissociate. The denaturation temperature of siRNAs depends on their length and composition. On-column denaturation can be observed by a peak shift since oligonucleotide duplexes adhere stronger to the stationary phase sothat single-strands elute earlier. Therefore, it is useful todetermine the denaturation temperature of the RNA sample.
Of course, LC can help to monitor the transition.

O

B1
O
-

P O

O

1. Choosing the optimum column temperature

has a negative influence on recovery and peak shape.
This effect is even more critical when working at low toneutral pH, as metals are more electropositive under these
conditions.
To overcome this problem HPLC systems and columns
can be passivated with strong acids or pre-conditioned
with a similar sample. However, these procedures are time
consuming and a recurring task. Furthermore, a change ofsample can lead to non-specific adsorption again.

The conventionally used materials for tubing and column
hardware pose a special challenge for the analysis ofoligonucleotides. Stainless steel provides mechanicalresilience and compatibility with most solvents althoughmany eluents such as methanol or acetonitrile can cause
corrosion. The resulting positively charged surface can leadto metal leaching as well as undesired ionic interactions withthe analytes. Electron rich analytes such as oligonucleotides
can be irreversibly adsorbed. This non-specific adsorptionOO

sense strand
antisense strand

S

Phosphodiester Linkage(All PO)

Name

O

HO

B2

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.de · www.ymc.eu

B1
H

P O
O

O

O

B2
H

B1, B2, …= thymine
Figure 1: Structure of poly(dT) oligonucleotides with thymine as bases.

Sample concentration
(μM)

194

25.7

25.7

2,980

2.0

2.0

6.0

15

4,501

1.0

1.0

4.5

dT 20mer

20

6,022

1.0

1.0

6.0

X = -H (DNA), -OH (RNA)dT 25mer25

dT 30meramine (TEA) is frequently used as the ion pair agent and isoften combined with the acidic counterion 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and methanol as organic modifier. The advantage of this mobile phase composition is its
compatibility with electrospray ionisation mass spectrometry (ESI-MS). This is based on the higher volatility of
HFIP, which results in faster evaporation of HFIP compared to TEA during ESI. The pH in the droplets increases and the TEA-paired oligonucleotide dissociates in thesource so that the detectability is increased.

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.de · www.ymc.eu

Length(mer)

Loading

Molecular weight
(Da)

–
10

dT 15mer

1. Effect of ion pair reagents and pH on oligonucleotide separation*1,1,1,3,3,3-hexafluoro-2-propanol

O

O
-

O

dT 10mer

Phosphorothioate Linkage(All PS)

Chromatographic separation of charged biomolecules by
RP is challenging because they are not retained sufficiently on the column. Since oligonucleotides possess an ionic
structure, ion pairing reagents with an opposite charge
need to be added to the mobile phase. Their hydrophobic part of the agent bonds to the hydrophobic stationary phase while the charged part pairs with the analytes
from the mobile phase. This results in the oligonucleotides
being retained on the stationary phase and separated
by length, type and presence of modifications. Triethyl-O

Caffeine (I.S.)

X

Figure 1: RNAs with phosphodiester linkage (All PO) and with sulphur modified phosphorothioate linkage (All PS).

YMC-Accura Triart Bio C18 (1.9 µm, 30 nm), 50 x 2.1 mm ID
TA30SP9-05Q1PTC
A) 8 mM triethylamine - 100 mM HFIP* (pH 8)B) methanol
1%B/min (initial = 3%B)
0.42 mL/min
65 °C, 45 °C or 25 °C
UV at 260 nm
1 µl (5 nmol/mL)
siRNA duplex, sense strand, antisense strand

oligo deoxythymidine (dT)

Table 1: Attributes of poly(dT) oligomers and sample conditions used.

X

B2
X

In this technical note, YMC-Triart Bio C18 is comparedwith XBridge Oligonucleotide BEH C18 and the DNAPac
columns, which are claimed to be specially designed forthe separation of oligonucleotides.

Poly(dT) oligonucleotides are single stranded nucleotide
chains, which solely consist of thymine as bases. Due
to base pairing of thymine and adenine, poly(dT) oligonucleotides are able to capture polyadenylated messenger
RNA (mRNA). This mechanism is widely used duringmRNA extraction and purification, for instance in thecourse of complementary DNA (cDNA) synthesis or mRNA
vaccine manufacturing. Therefore, the need for high purity
oligonucleotides is of paramount importance. For analysisof oligonucleotides, ion pair reversed phase (IP-RP) highperformance liquid chromatography (HPLC) is the mostcommon method used. IP-RP relies on the ionic interaction
between the analyte and the ion-pair reagent. The lipophilicalkyl chain of the ion pair reagent has high affinity to
the stationary phase, which maximises the retention ofanalytes on the phase. Due to the high resolution of IP-RP
chromatography, oligonucleotides with only minimal size
differences can be separated.

P O
O

O

HO

B1

The best choice for poly(dT) oligonucleotide analysis– YMC-Triart Bio C18

7,543

30

(1 µL)
pmol

1.0

9,064

(1 µL)
ng

1.0

1.0

5.0

7.5

1.0

9.1

dT 40mer

40

12,106

0.5

0.5

6.1

dT 60mer

60

18,190

0.5

0.5

9.1

dT 80mer

80

24,2740.3

7.3

dT 100mer

100

30,358

0.3

0.3

9.1

dT 120mer

120

36,442

0.2

0.3

0.2

7.3

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu

Page 1/5

Page 1/5

Page 1/10

Page 1/3

HILIC analysis ofoligonucleotides using
different bioinert columns

How to choose the optimum
conditions for analysis ofdenatured or non-denatured
siRNA duplexes

How to influenceoligonucleotide analysis
by IP-RPThe best choice for poly(dT)oligonucleotides analysis YMC-Triart Bio C18

APP NOTESAPPLICATION NOTEAPPLICATION NOTEAPPLICATION NOTE

The Benefit of Scalability of YMC-Triart Prep
in Oligonucleotide Separations
Introduction

Introduction

The development of a preparative process typically consists of three steps:• Method development at analytical scale• Loadability studies• Final scale-up to the preparative process

T

To perform this process development as smoothly as possible several aspects have to be taken into account.
It is very important to determine the final process scale before each screening. If the final purification goesbeyond an analytical scale, the availability of stationary phases for preparative purposes must be clarified.

N

O

O
O

O

O

O

O

P

O

O

P

O

O

O

O

HN

HN

N

O

O

CH
3

CH
3

After the preparative availability has been clarified, thestationary phase screening begins. This application
example uses oligonucleotides to show how asuitable separation is determined step by step. The
challenge here is that classic reversed phase liquid
chromatography is not directly applicable due to theincompatibility of the highly polar oligonucleotides andthe non-polarity of the stationary phase modifications.
Therefore, ion-pair reversed-phase chromatography(IP-RP) is a targeted approach. Ion pair reagentsare used as intermediaries between the polar
oligonucleotides and the non-polar stationary phase.
Such ion-pairing reagents are typically alkyl sulfonates,
alkyl ammonium salts, or similar compounds such as
triethylamine (TEA).

H3 C

Oligonucleotide Separations Using Ion-Pairing
Reversed Phase Liquid Chromatography

H3 C

NH

C H3

NH

C H3

C H3

The adsorption model on the other hand is based on aninteraction between the ion pairing reagent and the stationary phase occurring first. In this case, the hydrophobic
alkyl chain of the ion pairing reagent would be attached tothe alkyl chains of the stationary phase modification, presenting its potentially ionic site to the target compounds
in the mobile phase. This mechanism may be interpreted
as a pseudo ion exchange surface, which combines theadvantages of ion exchange resins (separation characteristics) with those of RP-HPLC phases (greater efficiency,pressure stability).
The use of ion pairing chromatography is the new gold
standard for the separation of peptides, oligonucleotides
and other molecules with charged functional groups.
Facing different objectives, the IP-RP conditions can be
optimised for analytical separations or preparative scale
purifications.

Oligonucleotides are important in genetic testing,research and forensics. For quite some time now,
oligonucleotide-based approaches have been developed
for different pharmaceutical applications. To monitor the
temporal and dimensional spreading of oligonucleotides
or proteins, they can be coupled to enhanced green
fluorescent protein (EGFP).

EGFP has a molecular weight of 26.9 kDa and is a single
point mutation of the green fluorescent protein (GFP)
which was first derived from jelly fish Aequorea Victoria.
EGFP has improved spectral characteristics such as
higher stability and a shift of the major extinction from
395 nm to 488 nm while the major emission is kept at
507 nm, which makes EGFP more suitable for existing
spectral analyses.

OH

groups protect the amino group of the base. This is only
required for the nucleobases adenine, guanine and cytosine as they all have primary amino groups which other-as silencing RNA (siRNA), messenger RNA (mRNA) andwise would attach to another nucleotide. For thymine, thisantisense oligonucleotides (ASOs) are gaining more andprotecting group is not required since it only has a lessmore attention in current research. Phosphoramidites arereactive secondary amine group. The 5’-hydroxyl group of

the essential part of the chemical synthesis of oligonu-

the sugar moiety is protected by a dimethoxytrityl (DMT)

cleotides, short fragments of nucleotides and analogues.

group. Inhibiting side reactions at the 3’-hydroxyl groupTo prevent any side reactions on residual reactive sitesof the sugar moiety is performed by two groups: a diiso-

such as hydroxyl and amino groups during oligonucleotide

propylamino and a 2-cyanoethyl (CE) group are attachedsynthesis, these groups need to be protected. Therefore,

to the phosphate atom. Figure 1 shows the protecting

the highly reactive native nucleotides are modified with fourgroups using the example of an adenosine based phos-

different protecting groups. Benzoyl (Bz) or isobutyryl (iBu)phoramidite.

(R)

N

O

(R)

NH2
(Z)

Dimethoxytrityl (DMT)
group

N

O

CH3

O

CH
3

O

O

N

O

N

N
O
O

N

O

OH
O

O

Benzoyl group

HN

H3C

N
HN

HN

O
O

P

O

O

O

ynthetic oligonucleotides are extremely promising
candidates for biopharmaceuticals in a wide rangeof diseases. This is why nucleic acid therapeutics such

HO

O

O
P

O

H C
3

NH

CH
CH

H C
3

3

3

Comparison study – oligonucleotide separation with analytical and preparative RP phases
Together with one of the leading global pharmaceuticalcompanies worldwide, an IP-RP separation of a 20mer
oligonucleotide was tested with different particle
sizes in order to examine the scalability of YMC-Triart.

S

O

O

should be achieved for all particle sizes used: from theanalytical particle size to the preparative quality. Usinga fully scalable stationary phase will greatly facilitate
method development as reliable results are obtained
from analytical to preparative scale.

HPLC Analysis of Phosphoramidites using RP or NP conditions

O

In order to achieve rapid screening with the highest
possible resolution, the stationary phases are mostly
used in analytical format. In this case it is very
important to check the scalability of the particles.
This means that comparable separation behaviour

Maximum Flexibility with YMC-Triart:

N

CH
3

C H3

raditionally, reversed phase liquid chromatography wasincapable of retaining highly polar compounds due tothe non-polar nature of the stationary phase modifications
used. Ion pairing reversed phase chromatography (IP-RP)
is an alternative approach which uses ion pairing reagentsto act as a mediator between the polar target molecules
and the non-polar stationary phase. Such ion pairing reagents are typically alkylsulfonates, alkylammonium salts
or similar compounds such as triethylamine (TEA).
While the exact mechanisms of the interactions are still
under investigation, there are currently two proposedmodels that describe the interactions which occur andthese may well be co-existing in reality: The partition
model assumes that the ion pairing reagent and the target
compound interact first, forming an ion pair that is lesspolar in nature in comparison to the target substance byitself, and in turn undergoes retention interactions withthe stationary phase.

APPLICATION NOTE

Analysis of mRNA coupled to enhanced green
fluorescent protein by RP and IEX

Figure 1: structure of EGFP (1)

NH

O

CH3

C H3
C H3

The particles sizes used were 3 µm (YMC-Triart C18)
as commonly used for analytical applications, and
7 and 10 µm (YMC-Triart Prep C18-S) for preparative
purification methods.

Ion pair reversed phase liquid chromatography (IPRP) continues to be the gold standard method for thecharacterisation of oligonucleotides. Certainly theelectron-rich backbone of oligonucleotides and thetypical use of stainless steel columns and tubing cancause an irreversible adsorption. This occurs due to ionicinteractions with the positively charged metal oxide layerof the component’s surfaces.

This effect is even more critical when working at low toneutral pH as metals are more electropositive at these
conditions. To overcome these challenges bioinert
systems and columns such as the recently introduced
YMC-Accura Triart columns can be used. YMC-Accura
Triart columns have a bioinert coating on all surfaces,
including the frits, to prevent any unwanted ionicinteractions.

P
H3C
Diisopropylamino
group

H3C

N
CH3

O

N
2-Cyanoethyl group

Figure 1: Protecting groups of an adenosine based phosphoramidite.

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email: info@ymc.eu · www.ymc.eu

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu

YMC Europe GmbH · Schöttmannshof 19 · 46539 Dinslaken · Phone +49 2064 427-0 · Fax +49 2064 427-222 · Email info@ymc.eu · www.ymc.eu

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The Benefit of Scalabilityof YMC-Triart Prep in Oligonucleotide Separations

Oligonucleotide Separations
Using Ion-Pairing Reversed
Phase Liquid
Chromatography

Analysis of mRNA coupled toenhanced green fluorescentprotein by RP and IEXMaximum Flexibility with
YMC-Triart: HPLC Analysis
of Phosphoramidites using
RP or NP conditions

43

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