Oligonucleotide Purification

Preparative Anion Exchange Chromatography
for the Purification of DNA Oligonucleotides

Anton Posch,1 Franziska Kollmann,1 Sharon Bola,2 Khaled Mriziq,2 and Elizabeth Dreskin2
1
Bio-Rad Laboratories GmbH, Kapellenstr. 12, 85622 Feldkirchen, Germany
2
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Abstract

DNA oligonucleotides obtained by solid-phase synthesis contain incomplete or erroneous sequences that
require removal for the advancement of these molecules as therapeutics. DNA oligonucleotides have ahigh degree of negative charge and therefore can be separated according to length with anion exchange
chromatography. Using crude preparations of 20- and 21-mer oligonucleotides, we demonstrate theefficacy of the Bio-Rad Nuvia HP-Q Resin with a bead size of 50 µm for DNA oligonucleotides. Evaluatedkey variables during method development include pH, flow rate, dynamic binding capacity, gradient slope,and scalability. Both small- and large-scale experiments with a 5 ml and 25 ml column, respectively, gave
excellent yields in the range of 90% while maintaining the high purity (>97%) of the full-length products.
Introduction
Oligonucleotides play a fundamental role in gene regulation andare considered a promising novel class of biopharmaceuticals(Catani et al. 2020). In 1998, the first oligonucleotide (ON) drug
was approved by the U.S. Food and Drug Administration, leading
to a growing interest in the development of oligonucleotide-based
therapeutics. Oligonucleotide-based drug platforms (Roberts etal. 2020) comprise many types of nucleic acids for specific gene
modulation, including antisense oligonucleotides (ASO), shortinterfering RNA (siRNA), micro RNA (miRNA), aptamers, andmessenger RNA (mRNA). ASOs, which are short (15–25 bases),
single-stranded DNA oligonucleotides designed antisense to target
RNA (Vanhinsbergh 2020), represent an important therapeutic
subclass. They are usually manufactured by cyclic solid-phase
synthesis based on phosphoramidite chemistry. Although
individual cycling efficiency is high (>98%), a low percentage ofthe oligonucleotide chain does not grow to the desired full-length
product, leading to overall product yield reduction and many
structurally similar impurities (Enmark et al. 2020). ON-failed
sequences are typically caused by the deletion or addition of
one or more nucleotides called shortmers (n – x) or longmers(n + x). Other impurities, which are comprehensively discussed by
El Zahar and colleagues (El Zahar et al. 2018), may be producedby failures during deprotection and detritylation or are caused bydepurination, deamination, and adduct formation.

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To increase biostability and the therapeutic efficiency of ONs,
they are commonly chemically modified at multiple sites:
phosphodiester backbone, ribose sugar, and the heterocyclic bases.
Phosphorothioation is the most common chemical modification of
ASOs and many ASOs are fully phosphorothioated to be protectedfrom in vivo nuclease degradation.
It is anticipated that ONs will gain importance as therapeutic
agents, and their production requires a highly efficient downstream
processing strategy after synthesis. Chromatography holds a key
position in this workflow and ON purification is usually achieved by
a combination of several techniques. A very successful purification
approach for the large-scale production of ASOs is based on the
combination of hydrophobic interaction chromatography (HIC)
and anion exchange chromatography (AEX) (Gronke 2020). HIC
is usually applied for the initial purification of DMT (dimethoxytrityl)on material while AEX is performed on the HIC eluate after
detritylation as a second polishing step (Andrews et al. 2021). AEX
mainly separates ASOs based on charge differences, leading to alength-based elution order, permitting the removal of key impurities
exhibiting deletion sequences. However, with AEX is it challenging
to separate full-length products from n – 1 contaminants andrequires the careful evaluation of a given AEX resin for its
applicability in a preparative ON purification process.

Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

Here, we present the characterization of the high-performancestrong anion exchange resin Nuvia HP-Q for the purificationof single-stranded DNA oligonucleotides. Developed for theindustrial downstream purification of large biomolecules at fast
flow rates, Nuvia HP-Q Resin has technical characteristics suchas bead size, pH stability, and flow characteristics that make theresin a promising candidate for ON purification. Key variables weevaluated include pH, flow rate, dynamic binding capacity, gradientslope, and scalability.

ONs 1–4 were used during method development while ONs 1
and 2 were also used for large-scale purification experiments. For
chromatography, dried ONs were dissolved in buffers preparedusing nuclease-free water.
Chromatography

Equipment and resins used for chromatography experiments(Table 2) were from Bio-Rad Laboratories, Inc. Chromatography wasmonitored at 260 and 280 nm and performed at room temperatureusing an NGC 10 Medium Pressure Chromatography System. The
chromatography buffers were thoroughly degassed and filteredthrough 0.45 µm membranes. The four-tier chromatography system
was configured with a sample pump including a sample inlet valve,
two buffer inlet valves, a column switching valve, a buffer blending
valve, a multi-wavelength UV detector (5 mm path length), a pH
and conductivity meter, an outlet valve, and a fraction collector
with a benchtop Peltier cooling option. Chromatography operating
conditions are presented in Figure 1. Samples from analytical andpreparative runs were stored at 4°C and pH was not adjusted prior
to analysis by electrophoresis.

Materials and Methods*
Oligonucleotides

We used four custom-made (Sigma Aldrich) desalted, lyophilized
single-stranded DNA oligonucleotides (3 x 20-mer and 1 x 21-mer)with phosphodiester backbones (Table 1).
Table 1. DNA oligonucleotides used in this study. Name, length, and sequence
are listed.
Oligo Name

Length (nucleotides)

Sequence (5'–3')

Oligo 1 (ON 1)

20

ATA CCG ATT AAG CGA AGT TT

Oligo 2 (ON 2)

21

ATA CCG ATT AAG CGA AGT TTT

Oligo 3 (ON 3)

20

TAT GGC TAA TTC GCT TCA TC

Oligo 4 (ON 4)20

GCC CAA GCT GGC ATC CGT CA

Table 2. Overview of chromatography columns used for DNA oligonucleotide purification and fraction analysis, including buffer and operating conditions.
Column/Resin Type

Resin Type,
Bead Size

Column Dimensions,
Pack Type

Application

Buffer A

Buffer B

Foresight Nuvia HP-Q
(5 ml; catalog #12007021)

Strong AEX,
50 µm

8 x 100 mm, prepacked

Method development

25 mM arginine-NaOH, pH 11.7

25 mM arginine-NaOH,
2 M NaCl, pH 11.7

120–240

Nuvia HP-Q
(25 ml; catalog #12006693)

Strong AEX,
50 µm

25 x 100 mm, self-packed Large-scale
into Econo Alpha Column purification(#12009461)

25 mM arginine-NaOH, pH 11.7

25 mM arginine-NaOH,
2 M NaCl, pH 11.7

100–120

ENrich Q(1 ml; catalog #7800001)

Strong AEX,
10 µm

5 x 50 mm, prepacked

25 mM arginine-NaOH, pH 11.7

25 mM arginine-NaOH,
2 M NaCl, pH 11.7

300

Nuvia HP-Q (5 and 25 ml)

ENrich Q (1 ml)

Equilibration
5 CV with 2% buffer B

Equilibration
5 CV with 20% buffer B

Sample application
ON (0.1 to 0.3 mg/mL) in 2% buffer B

Sample application
Sample in 20% buffer B

Postapplication wash
5 CV with 2% buffer B

Postapplication wash
2 CV with 20% buffer B

Gradient elution 1
15 CV with X*–23% buffer B

Gradient elution
20 CV with 20-35% buffer B

Gradient elution 2
40 CV with 23–35% buffer B

Column cleaning
5 CV with 100% buffer B

Fraction analysis

Flow Rate,
cm/hr

Gradient elution 3
5 CV with 35–50% buffer B
Column cleaning
5 CV with 100% buffer B© 2021 Bio-Rad Laboratories, Inc.

Fig. 1. Chromatography operating conditions. Proposed phases for the preparative purification of DNA oligonucleotides (ONs)with Nuvia HP-Q Resin and subsequent fraction analysis with analytical anion exchange chromatography (Enrich Q Resin). CV,
column volumes. X,* conductivity conditions at elution start are ON-specific and are optimized individually.

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Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

Electrophoresis

Optimization of Media, pH, and Flow Rate

The quality of the chromatographic purification experiments ingeneral and ON length–based purity were monitored by 15%
midsize Criterion TBE-Urea Polyacrylamide Gels available withdifferent well numbers (12 wells, #3450091; 18 wells, #3450092;
26 wells, #3450093) in conjunction with 1x Tris/Boric Acid/EDTA(TBE) running buffer (#1610770). Samples for analysis (typicalconcentration range: 20–200 ng/µl) were dissolved 1:1 in 2x TBE
urea sample buffer (Alfa Aesar, #J60186), which consists of
90 mM Tris base, 90 mM boric acid, 2 mM EDTA, 12% Ficol
Solution, 7 M urea, 0.03% bromophenol blue, and 0.03% xylene
cyanole. Samples were heated at 70°C for 4 min and the preferredloading volume was 10 µl. After sample loading, gels were run
at constant mA until the bromophenol blue front reached
two-thirds of the gel length (for example, 30 min at 10 mA and
30–40 min at 20 mA). After the run, the gels were washed 2x for
2 min in water and stained in the dark for 30 min with 1x
polyacrylamide gel electrophoresis (PAGE) GelRed Nucleic Acid
Gel Stain (Biotium, Inc., #41003) dissolved in electrophoresis
buffer or water. The stained gel was washed again in the darkwith water for 2x for 2 min and imaged with the GelDoc Go Gel
Imaging System (#12009077) using the preset acquisition settingsfor ethidium bromide. Gel analysis was performed with ImageLab
software (version 6.1.0).

Single-stranded DNA oligonucleotides are relatively stablemolecules at slightly alkaline conditions and are negatively charged
at physiological pH and above. AEX chromatography separates
ONs by their chain length and high-resolution separations can beobtained for up to 40-mers, as demonstrated with the Bio-Rad UNO
Q Resin with a continuous-bed matrix (Bio-Rad Laboratories 1998).
Resin selectivity in AEX is influenced by a variety of parameters,
including bead size, buffer composition (pH, molarity, ion type),
column dimensions, gradient slope, flow rate, temperature, andorganic modifiers. Usually, bead size for resins applied in ON
purification are in the range of 20–45 µm and successful ON
purification schemes using AEX have been reported at different
pH values (7.5–8, 9.0–9.5, 12), indicating the importance of pH
optimization during method development. High pH values provide
denaturing conditions and eliminate the negative effects of theONs’ secondary structure and self-assembly by hydrogen bonding.
On the other hand, a few AEX resins do not show long-term
stability at elevated pH levels and the selection of a buffer systemfrom pH 8–10 is more appropriate. Although higher separation
temperatures (>70°C) and the addition of organic modifiers likemethanol might be beneficial for ON purification, these measures
add additional cost to a commercial production process and areusually not considered further.

Analytical Assays

In early experiments, two strong AEX resins (Macro-Prep 25 Q and
Nuvia HP-Q) were considered for ON purification since they aredesigned with an optimal bead size to achieve both laboratory- andprocess-scale purification of biomolecules at high flow rates without
being limited by column pressure. The Macro-Prep 25 Q material has
the advantage of a smaller bead size (25 µm vs. 50 µm), increasing
peak resolution, but has limited alkaline stability of pHmax = 10.

For rapid ON quantification, 2 µl of sample were applied toa NanoQuant Plate (Tecan Trading AG, #30033939) andabsorbance at 260 nm was measured with an Infinite 200 Plate
Reader (Tecan, #30016056).
The fraction of the full-length target ON in contaminated samples
or side fractions obtained after chromatographic purification were
measured after further separation with a high-resolution analytical
AEX column (ENrich Q 5 x 50 mm, #7800001). The analytical AEX
column can separate the full-length target ON from its contaminants(n – x and n + x). Peak integration and quantification were done with
ChromLab Software (version 6.1.27).

As test samples, ON 1 and 2, which differ by a single nucleotide
in length, were mixed at a ratio of about 1:1 and analyzed withboth resins at each of two different pH values. Figure 2 shows theperformance of Macro-Prep 25 Q Resin at pH 8 and 9.5 and Nuvia
HP-Q Resin at pH 9.5 and 11.7. With the Macro-Prep 25 Q Resin,no distinct separation of the two ONs was obtained at either pH.
The Nuvia HP-Q Resin runs were successful at pH 11.7, showing aclear separation of the two ONs, but at pH 9.5 a profile with largely
overlapping ONs was recorded.

* Materials, instruments, and software from Bio-Rad Laboratories, unless otherwise
noted.

Results and Discussion
Due to the high complexity of contaminants obtained after thechemical synthesis of single-stranded oligonucleotides, a carefulanalysis and optimization of critical variables in AEX purification isnecessary (Deshmukh et al. 2000). Deshmukh and colleagues
describe an excellent framework for this task and provide universal
findings, which can be used to shorten the characterization process
for resins considered for application in this specific workflow. Basedon this process, our characterization and optimization strategy was
restricted to the effect of pH, flow rate, dynamic binding capacity,and gradient slope accompanied by an analysis strategy basedon two complementary techniques: denaturing PAGE andanalytical AEX.

© 2021 Bio-Rad Laboratories, Inc.

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Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

A
400
400

250
250

300
300

200
200
150
150

30
30
200
20
20

00

10
10

12
12

14
14

16
18
16
18
Column volumes, CV

20
20

12
12

Volume, ml

14
14

16
16

18
18

Columnvolumes,Volume,mlCV

Oligo 1

600
600

22
22

Oligo 3

20
20

22
22

Oligo 4λ260
λ260,, mAU
mAU

250
250
200
200

40
40

400
400

30
30

300
300
20
20

200
200

10
10

100
100
00

150
150

10
10
pH 9.5

100
100

00

50
50

500
500

300
300

260

10
10

B

B

λ260
λ , , mAU
mAU

10
10

pH 9.550
50

pH 11.5

12
12

14
14

16
16

18
18

Columnvolumes,Volume,
mlCV

20
20

22
22

00

Fig. 3. Influence of eluent pH on retention time. Separation profiles of three
Oligo 1(ON 1, —;
Oligo
Oligo
4 4, —) at A, pH 9.5
different 20-mer oligonucleotides
ON33, —; andON
and B, 11.7 on a 5 ml Nuvia HP-Q Column. The increase in eluent pH results in a
retention shift toward higher conductivity conditions and less peak fronting.

50
50

00

40
40

1008
pH
100

100
100

00

50
50

%B
%B

λ 260
λ260,, mAU
mAU

300
300

%B
%B

260

λ , , mAU
mAU
λ 260

A

10
10

12
12

14
14

1616
1818
Column volumes, CV

2020

2222

The effect of flow rate in preparative AEX on both productivity
and peak purity is diametrical and needs to be balanced to serveboth needs; it is also probably sample dependent. The
5 ml Nuvia HP-Q Column was tested at two flow rates (120 and
240 cm/hr) and the corresponding results for a 1:1 mixture of
ONs 1 and 2 are shown in Figure 4. As expected, the lower flowrate shows a superior resolution. Although separation quality at
240 cm/hr showed an increase of the overlapping zone, this flowrate might still be sufficient for the removal of short and longmers
at an acceptable yield. Reported flow rate values from competitor
resins with smaller bead sizes are in the range of 150–220 cm/hr.
In our case, the experiments for dynamic binding and largescale purification were all conducted at 100–120 cm/hr, because
separation quality and thus improved yield were higher valued thangain in process speed.

Volume, ml

Fig. 2. pH-dependent resin performance testing. Optimization was done at a flowrate of 120 cm/hr with a 1:1 mixture of ON 1 and 2. A, Macro-Prep 25 Q Resin at
pH 8, (—) and 9.5, (—); elution conditions: 20–35% buffer B (20 CV). B, Nuvia HP-Q
Resin at pH 9.5, (—) and 11.7, (—); elution conditions: 20–40% buffer B (20 CV). pH
8 and 9.5 buffers are Tris-based (25 mM) instead of arginine-NaOH (see Table 2).
Column volume was 5 ml.

The obvious pH-dependency of peak shape and resolution was
further evaluated with three 20-mer oligonucleotides that differ
significantly in nucleotide sequence or composition (see Methods
section). ONs 1, 3, and 4 were chromatographed individuallyat pH 9.5 and 11.7 and the results are presented in Figure 3.
Peak fronting is obvious at pH 9.5 for all three samples, but highquality profiles were obtained at pH 11.7. Similar observations
have been published by Deshmukh et al., who reported that ON
separations with pH conditions between 11 and 12 show dramatic
improvement in peak resolution.

© 2021 Bio-Rad Laboratories, Inc.

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Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

7 M urea. Here, denaturing PAGE was chosen because up to
26 fractions can be analyzed on a single gel and the results are
obtained within 2.5 hours. It is important to mention that samples
considered for electrophoresis should be adjusted with water or
chromatography buffer to obtain similar conductivity conditions acrossall samples. This adjustment step can be omitted by extending the120entry
cm/hstep at 10 mA constant/gel from 30 to 50 min.
sample300
250

λ260, mAU

150

240 cm/h

100

After fraction screening with denaturing PAGE, it is recommended togenerate a minimum of three fraction pools for further analysis, forexample, pool A, ON with n – x contaminants; pool B, ON, highly
enriched full-length product; pool C, ON with n + x contaminants.

50
0
12
12

14
14

16
18
16
18
Column volumes, CV

20
20

22
22

The second component of our analysis strategy was achromatography-based quantitative analysis of fraction pools A
and C. Using an analytical 1 ml ENrich Q Column, we calculated
the ON material lost during the preparative purification process.
Analysis of pool B was necessary for assessing product purity.

Volume, ml

Fig. 4. Influence of flow rate on separation quality. Separation profile of a 1:1
mixture of ON 1 and 2 using a 5 ml Nuvia HP-Q Resin at pH 11.7 with two different
flow rates: 120 cm/hr (—) and 240 cm/hr (—). Sample: 1:1 mix of ON 1 and 2.

Dynamic Binding Capacity of Nuvia HP-Q

To understand the nature of and to quantify contaminating species
present in ON 1 and 2 raw material and to test the performanceof our gel analysis system (15% TBE-urea PAGE), a 1:1 mixtureof both ONs (each 2 mg) was generated and purified with a 5 ml
Nuvia HP-Q Column (Figure 6A). The chromatogram was divided
into seven sections (A–G) and both individual and pooled fractions
were analyzed with denaturing PAGE (Figure 6B).

To assess the dynamic binding capacity of the Nuvia HP-Q Resin,
60 mg of lyophilized ON 1 was first dissolved in 275 ml binding
buffer (25 mM arginine-NaOH, 40 mM NaCl, pH 11.7), resulting in aconcentration of 0.18 µg/µl at 260 nm. This solution was then applied
to determine the dynamic binding capacity at 10% breakthroughwith a flow rate of 120 cm/hr. Figure 5 shows the breakthroughcurve for a 1 ml Nuvia HP-Q Column with a measured dynamic
binding capacity of about 20 mg crude ON 1 per ml of resin.

A
3,000

3,000
3,000
A max

λλ260
, mAU
260, mAU

λ 260
λ260,, mAU
mAU

2,500
2,500
2,000
2,000
1,500
1,500

B

C

D

E

F

110

120

G

2,000
1,500
1,500
1,000
1,000
500
0

1,000
1,000

30

30

500
500
0

A

2,500

10% of A max

10
10

30
30

50
50

70
70

90
90

110
110

130
130

B

100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
00

40

40

50

50

60

60

70

70

80

80

90

100

90
100
Volume, ml

Volume, ml

110

120

130

130

140

140

150

%B

10
10

%B

λ260, mAU

200

150

C

150
150

Volume,ml
Elutionvolume,ml

Oligo 1,raw

Fig. 5. Dynamic binding capacity of Nuvia HP-Q Resin at 10% breakthrough.
Using ON 1 (0.18 mg/ml), binding capacity was determined to be 20 mg
oligonucleotide raw material per ml resin. Flow rate: 120 cm/hr.

Oligo 1,
purified,pool C

Analysis Strategy for Determination of Fraction Pooling, Yield, and Purity

Fraction pooling, yield, and purity calculations require a proper
analysis strategy. After ON purification, fractions that are significantly
contaminated with n – x and n + x species must be identified andexcluded from the fraction pooling process to obtain the full-length ON
at a desired purity. Analytical methods for successful fraction purity
analysis include capillary electrophoresis, high performance liquid
chromatography (HPLC), AEX, or size exclusion chromatography(SEC) columns, and denaturing electrophoresis with polyacrylamidegels containing© 2021 Bio-Rad Laboratories, Inc.

Fig. 6. Separation of individual ONs from contaminating species to assess
analysis strategy. A, Separation of each 2 mg ON 1 and ON 2 with a 5 ml Nuvia HP-Q
Column at pH 11.7 and 120 cm/hr. Section A: fraction pool, elution volume 34–85 ml,
concentrated with a 1 ml Nuvia HP-Q Column; section B: covers four individual 1 ml
fractions: B1–B4, elution volume 86–89 ml; section C: fraction pool, elution volume
90–98 ml; section D: covers six individual 1 ml fractions: D1–D6, elution volume
99–104 ml; section E: fraction pool, elution volume 105–113 ml; section F: covers fiveindividual 1 ml fractions: F1–F5, elution volume 114–118 ml; section G: fraction pool,
elution volume 119–132 ml. B, fractions from sections A–G were further analyzed
with denaturing PAGE. C, purity comparison of enriched ON 1 (—, fraction pool,
Section C) and ON 1 raw material (—) by analytical AEX.

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Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

Fractions containing n – x or n + x species in the presence offull-length ON 1 were easily identified with denaturing PAGE andpooled afterward, as described above (data not shown). Pool A
had a volume of 6 ml, pool B with purified ON 1 had 172 ml, andpool C had 40 ml. The three samples, together with the starting
material and a 1:1 mixture of ON 1 and ON 2, were applied todenaturing PAGE (Figure 8) and assessed for purity. Additionally,
fraction pools A, B, and C were analyzed with a 1 ml ENrich Q
Column and quantified for ON 1 content (data not shown).

The two components of the ON sample mixture appeared as two
individual bands on the gel image, indicating the high resolutionof the 15% TBE-urea gel system and its suitability to separate afull-length ON from its n – x and n + x contaminants (as can beseen in fractions B – 3 and B – 4 for the n – x species). Fraction
pool C contains highly enriched ON 1, while fractions D1–D6
visualize the transition from ON 1 to ON 2 according to the Nuvia
HP-Q chromatogram (Figure 6A). Very pure ON 2 product can befound in fractions E and F1–F5, while in fraction pool G, ON 2 iscontaminated by a clearly visible 22-mer.
The purity of fraction pool C, which contains highly enriched
ON 1, was analyzed with a 1 ml ENrich Q Column and compared
to unpurified material. As can be seen in Figure 6C, pool C appears
as a very homogeneous peak without any of the by-products
observed in the unpurified sample.

1

2

3

4

5

ON 2, MW = 6,444 Da
ON 1, MW = 6,140 Da

The data obtained from this small-scale ON purification suggestthat the proposed analysis strategy for fraction pooling andproduct quality analysis based on denaturing PAGE and analytical
AEX chromatography can be very well integrated into large-scale
purification experiments for single ONs.
Purification of ON 1 Using a 5 ml Column

Lyophilized, unpurified ON 1 was dissolved in binding buffer toreach a concentration of ~0.1 mg/ml at 260 nm and 724 ml ofsample solution was applied to purification with a 5 ml Nuvia HP-Q
Column at 120 cm/hr. The sample solution contained about 56 mg
full-length ON 1. The corresponding chromatogram is displayed in
Figure 7. Due to the high concentration of ON 1 in the eluate, thedetection at 260 nm reached its limits in combination with a 0.5 cm
flow cell.
4,500
4,500

A

4,000
4,000

B

90
90
80
80
70
70

3,000
3,000

%B

50
50

2,000
2,000

%B

60
60

2,500
2,500

260

λ 260
mAU
λ ,, mAU

The evaluation of the various datasets revealed an ON 1 purity of≥96% and the yield of the preparative chromatographic run was
calculated to be ~90%. A higher purity level can be easily obtainedby changing the pooling strategy based on denaturing PAGE resultsby excluding additional fractions, either pre– or post–main peak.

100
100

C

3,500
3,500

40
40

1,500
1,500

30
30

1,000
1,000

20
20

500
500

0

Fig. 8. Denaturing PAGE analysis of fraction pools obtained after the
purification of ON 1. Fractions were obtained by AEX chromatography with a 5 ml
Nuvia HP-Q Column. Lane 1, 1:1 mixture of ON 1 (6,140 Da) and ON 2 (6,444 Da);
lane 2, unpurified ON 1; lane 3, pool A; lane 4, purified ON 1, pool B; lane 5, pool C.

1010

10–23%B

750
750

800
800

850
850

900
900

950
950

1000
1,000

1050
1,050

00

The applied elution scheme worked very well for ON 1, but weassume that elution segment 1 needs to be adjusted for other typesof ONs, especially for n-mers smaller than 20, because elution
segment 1 starts with a NaCl concentration of 200 mM (10% B)
to concentrate n – x species at a comparable volume as used forON 1. A more universal approach, such as gradient elution from40 to 800 mM salt over 50 column volumes, is a good starting point,
but was not verified in practice and depends on the nature andamount of ON to be purified.

1100
1,100

Volume, mlml
Volume,Fig. 7. ON 1 purification with a 5 ml column. Purification of about 72 mg ON 1 raw
material with a 5 ml Nuvia HP-Q Column at pH 11.7 and 120 cm/hr. Fraction pool B
contains highly enriched ON 1 with a purity of ≥96% at a process yield of 90%.

© 2021 Bio-Rad Laboratories, Inc.

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Preparative Anion Exchange Cchromatography for the Purification of DNA Oligonucleotides

Separating Purified ON 2 “Contaminated” with Purified ON 1

The separation quality of n – 1 from n was analyzed in more detailwith a defined artificial sample (ON 1:ON 2 ratio of 1:15) by using
purified ONs. Purified ON 1 (3 mg) was spiked into 45 mg purified
ON 2. Additionally, elution gradient segment 1 was changed from
10–23% B to 2–23% B to evaluate the distribution width of
ON 1 (here, our contaminant) by applying a smoother transitionof conductivity conditions after the sample application step. The
corresponding chromatogram is displayed in Figure 9 and the first18 ml of the elution peak were analyzed with denaturing PAGE
(Figure 10).
A

4,000
4,000

90
90

3,500
3,500

80
80

45

Pool A1

1.1

0

Pool A2

2.1

2

Pool A3

0

1

Pool B

0

41

Starting
material

Zone A1

Starting material

Fig. 11. Analytical AEX chromatography for the characterization of different
samples obtained after Nuvia HP-Q purification (see Figure 9) of an artificial
ON sample (ON 1:ON 2 ratio = 1:15). The shoulder to the left of the main peak forthe starting material is the ON 1 “contamination” in the sample.

1

2

3

4

5

6

ON 2, MW = 6,444 Da
ON 1, MW = 6,140 Da

%B

50
50

2,000
2,000

%B

60
60

2,500
2,500

40
40

1,500
1,500

30
30

1,000
1,000

00
500
500

3

Zone A2

70
70

3,000
3,000

500
500

mg Oligo2

Sample100
100

B

λ260, mAU

λ260, mAU

4,500
4,500

mg Oligo1

Zone A32020

2–23%B

550
550

Fig. 12. Assessment of ON 2 purity and yield. Denaturing PAGE analysis of fraction
pools obtained after the purification of an artificial ON sample (ON 1:ON 2 ratio = 1:15)
by AEX chromatography with a 5 ml Nuvia HP-Q Column. Lane 1, 1:1 mixture of ON 1
and ON 2; lane 2, sample with ON 1:ON 2 ratio = 1:15; lane 3, pool A1; lane 4, pool A2;
lane 5, pool A3; lane 6, pool B.

10
10

600
600

650
650

700
700

750
750

800
800

850
850

00

900
900

Volume, ml
Volume,mlFig. 9. Separation of purified ON 2 spiked with purified ON 1. Purification ofan artificial mixture of both purified ON 1 and ON 2 (ratio 1:15) with a 5 ml
Nuvia HP-Q Column.
A1, 7 ml

A2, 7 ml

The change in elution strategy for segment 1 seems to be beneficial,since a significant portion of ON 1 was collected as pure fractions incomparison to the dataset presented previously, although the widthof the contamination zone A2 with both ONs present is similar to theresults reported above for ON 1 purification with the 5ml column.

A3, 4 ml

Purification of ON 1 Using a 25 ml Column

For large-scale purification of ON 1, an Econo Alpha Glass Column(25 x 100 mm) was used to pack a 25 ml Nuvia HP-Q Column at aflow rate of 120 cm/hr and later operated at 100 cm/hr. Lyophilized,
unpurified ON 1 (new batch) was dissolved in binding buffer to reachconcentration of ~0.23 mg/ml at 260 nm and 1,740 ml of sample
solution (400 mg raw material) was applied to a fivefold scale-up
purification run by using the optimized gradient slope described forthe spiked experiments above. In comparison to the 5 ml column(8 x 100 mm) used for method optimization, the bed height of the
Nuvia HP-Q Resin in the preparative Econo Alpha Column is about
50% shorter, while its diameter is increased by a factor of three.
The corresponding AEX chromatogram is displayed in Figure 13
and fractions were analyzed with denaturing PAGE to identify the
distribution of ON 1 across a 1,500 ml elution volume.

Fig. 10. Analysis of the first eluent peak with denaturing PAGE. Denaturing PAGE
analysis of 18 individual fractions of section A from purified ON 2 spiked with ON 1.

The first seven 1 ml fractions contained highly enriched ON 1(pool A1), the next seven 1 ml fractions (pool A2) contained mixtures
where both ONs were present at different ratios, and the final 4
ml (pool A3) was dominated by ON 2. Fraction pools A1–A3 were
further analyzed with ENrich Resin and compared against the artificial
sample (Figure 11). Data analysis revealed 97% and 93% ON 2 purity
and yield, respectively. Finally, Figure 12 shows an overview gel withall relevant samples under investigation.

© 2021 Bio-Rad Laboratories, Inc.

7

Bulletin 3290

Preparative Anion Exchange Chromatography for the Purification of DNA Oligonucleotides

4,500
A1 A2

4,000

B

C1

C2

C3

90
90

3,500

80
80

3,000

60
60

2,000

50
50

1,500
1,500

40
40
30
30

1,000
1,000
500
0

%B

70
70

2,500

%B

260

λ260λ, mAU
, mAU

100
100

20
20
10
10

2–23%B
65
1,800

75
2,100

85
2,400

95
105
2,700
3,000
Volume,Volume, mlml

115
3,300

125
3,600

00

Fig. 13. Large-scale purification of ON 1. About 400 mg raw ON 1 was purified
using a 25 ml Nuvia HP-Q Column at pH 11.7 and a flow rate of 100 cm/h. Fraction
pool B contains highly enriched ON 1 with a purity of 97% at a process yield of 90%.

Six fraction pools (A1, A2, B, C1, C2, and C3) were generated
for further yield and purity analysis based on analytical AEX
chromatography (data not shown) and denaturing PAGE (Figure 14).
Pool B contains highly purified ON 1, while in pools A2 and C1,
ON 1 is contaminated with either n – x or n + x oligo species.
Fraction pools A1, C2, and C3 are solely composed of contaminating
species without ON 1. Data analysis of fraction pools A2, B, and C1
revealed an ON 1 content of 25 mg, 297 mg, and 6 mg, respectively.
In summary, the total yield for ON 1 at a purity of > 97% is ~90%
and very comparable to the results obtained by the small-scale
analytical 5 ml HP-Q Column. Together, the data suggest a
successful scale-up strategy.
1

2

3

4

5

6

7

Concluding Remarks
The production of pure single-stranded DNA oligonucleotides
requires efficient, scalable, and cost-effective chromatographic
purification methods applied after chemical synthesis. Key
impurities in crude samples are length based and are best
removed by AEX chromatography. Here, we show that Nuvia HP-Q
Resin is well-suited for ON purification, as demonstrated withtwo crude ON species (20-mer and 21-mer) with phosphodiester
backbones. Key process parameters like flow rate, gradient slope,buffer pH, and dynamic binding capacity were explored with aprepacked 5 ml column and the resulting protocol optimized foryield was successfully applied to a self-packed 25 ml column.
Similar yield (~90%) and purity (~97%) data were obtained withboth column sizes and strongly indicate the scalability of themethod, although not tested at pilot scale. We also suggest that
Nuvia HP-Q is a promising resin for the efficient purification of fully
thioated oligonucleotides used in clinical trials.
References ndrews BI et al. (2021). Sustainability challenges and opportunities inAoligonucleotide manufacturing. J Org Chem 86, 49–61.
 io-Rad Laboratories (1998). Nucleotide and oligonucleotide separations on an UNO
B
Q1 ion exchange column. Bio-Rad Bulletin 2352.
Catani M et al. (2020). Oligonucleotides: Current trends and innovative applications inthe synthesis, characterization, and purification. Biotechnol J 15, e1900226.
Deshmukh RR et al. (2000). Process development for purification of therapeutic
antisense oligonucleotides by anion-exchange chromatography. Org Process Res
Dev 4, 205–213
El Zahar NM et al. (2018). Chromatographic approaches for the characterization andquality control of therapeutic oligonucleotide impurities. Biomed Chromatogr 32,
Epub.
Enmark M et al. (2020). Analytical and preparative separation of phosphorothioatedoligonucleotides: Columns and ion-pair reagents. Anal Bioanal Chem 412, 299–309.
Gronke R (2020). Antisense oligonucleotide purification process: Successes andchallenges during scale-up. https://biopharma-asia.com/webinars/antisenseoligonucleotide-purification-process-successes-and-challenges-during-scaleup/#webinar_embed, accessed December 22, 2021.
 oberts TC et al. (2020). Advances in oligonucleotide drug delivery. Nat Rev Drug
R
Discov 19, 673–694.

ON 1, MW = 6,140 Da

Vanhinsbergh CJ (2020). Analytical Separation Methods for Therapeutic
Oligonucleotides. Advances in Biopharmaceutical Analysis 33, 20–26.

Visit bio-rad.com/OligoPurification for more information.
Fig. 14. Denaturing PAGE analysis of fraction pools obtained after large-scale
purification of ON 1. Fraction pools were obtained by AEX chromatography with a
25 ml Nuvia HP-Q Column. Lane 1, starting material; lane 2, pool A1; lane 3, pool A2;
lane 4, pool B (purified ON 1); lane 5, pool C1; lane 6, pool C2; lane 7, pool C3.

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