GX-271 Oligo Purification System:
Streamlined Purification ofAssay‑Ready Oligonucleotides byAutomated HPLCAPPLICATION NOTE AN1008

APPLICATION BENEFITSSOLUTIONS

Custom manufactured oligonucleotides require additional
purification steps after synthesis to separate the full‑length
products from contaminants such as truncated sequences, baseprotecting groups, and other impurities. Efficient purificationof oligonucleotides, of different lengths and at different
production scales, presents a manufacturing challenge.

The GX-271 Oligo Purification System, configured with
automated preparative and analytical HPLC capabilities,
can be used to isolate, analyze, and desalt reagent-gradeoligonucleotides in one continuous run. On-board fraction
analysis during runs enables on‑the-fly sample addition
resulting in no downtime on the instrument. Built-in error
handling conditions at various stages of the process prevent
sample loss and assure full walk‑away capabilities.

LAINE STEWART, LUKE ROENNEBURG, AND KAREN KLEMAN | GILSON, INC., MIDDLETON, WICHRIS SHERRILL | LUMINEX CORP., MADISON, WI

ABSTRACT“…a single liquid handling platform […]Oligonucleotides must be of high purity
when used in molecular biology applications,but efficient purification over a large rangeof production scales is challenging. TheGilson GX-271 Oligonucleotide Purification Systemprovides a versatile solution for continuous
separation, analysis, and desalting ofoligonucleotides.

used to isolate, analyze, and desalt
reagent-grade oligonucleotides in
one continuous run.”

INTRODUCTION
Oligonucleotides are crucial components in many
molecular biology techniques, such as qPCR,
next generation sequencing (NGS), and microarray
analysis and also have applications as therapeutics.1
These custom manufactured compounds require
additional purification steps after large scale
synthesis. The full-length products must be
separated from truncated sequences, inhibiting salts,
and base protecting groups. Efficient purification ofoligonucleotides, of different lengths and at different
production scales, presents a manufacturing
challenge.
To overcome these challenges, Gilson has developed
a single liquid handling platform configured
with automated preparative and analytical HPLC
capabilities that can be used to isolate, analyze,
and desalt reagent-grade oligonucleotides in one
continuous run.

Figure 1

GX-271 Oligo Purification System

MATERIALS AND METHODSANALYTICAL

All solvents used for chromatography were HPLC
grade. Gilson TRILUTION® LC software was used
to control instrumentation during the purification
process.
Crude synthetic oligonucleotides were injected ontoa preparative ion-exchange column. Small samples of
selected column fractions were reinjected to confirm
identity. Fractions with established identities weredesalted with a size-exclusion desalting column.
Samples and Reagents
Crude synthetic oligonucleotides were used as-is
following laboratory synthesis.
MultiCode®
Base PairH

Liquid Handler

GX-271 Liquid Handler with
Two GX Direct Injection Modules

Mobile Phase Pumps

322 HPLC Pump with H2 Heads

Detector

159 UV-VIS Detector with
10 mm Flow Cell

Switching Valves

VALVEMATE II Valve Actuators

DESALTING
Liquid Handler

GX-271 Liquid Handler with
Two GX Direct Injection Modules

Mobile Phase Pumps

322 HPLC Pump with H2 Heads
VERITY® 1810 Conductivity and pHMonitor

NNNH

Deoxyribose

Detector
159 UV-VIS Detector with0.5 mm Flow Cell

iGNO

NH

Switching Valves

ON

VALVEMATE II Valve Actuators

iCHNN

Deoxyribose

H

Chromatography Methods
PREPARATIVE METHODNColumnNO
HDeoxyribose

MobilePhase

GHNNNHHNH

B = A + 1M NaBr

9.95–19.9 min: 100% A to 100% B

O

19.9–20 min: 10% A/90% B

Standard
Base Pair

Deoxyribose

A = 20% ACN, NaPO4, pH8.5

0–9.95 min: 100% A

NN
CTosoh SuperQ Column, 10 x 200 mmFigure 220–119.90 min: 10% A/90% B to 35% A/65%B
GradientLuminex MultiCode® Technology119.9–120 min: 35% A/65% B
120–130 min: 35% A/65%B to 100% B
130–140 min: 100% B

Instrumentation

140–141 min: 100% B to 100% A

PREPARATIVE

141–155 min: 100% A

Liquid Handler

GX-271 Liquid Handler with
Two GX Direct Injection Modules

Flow Rate

5 mL/min

Mobile Phase Pumps

322 HPLC Pump with H2 Heads

Injection
VolumeVariable; set at run time

Detector

159 UV/VIS Detector with
0.5 mm Flow Cell

Switching Valves

VALVEMATE® II Valve Actuators

Fraction
Collection
Settings

Conditional fraction collection by peak level;variable; set at run time

2/4ANALYTICAL METHOD
ColumnMobilePhase

Dionex DNAPac PA-200
A = 20% ACN, NaPO4, pH11
B = A + 1M NaBr
0–2.90 min: 15% A/85% B
2.9–13 min: 15% A/85% B to 50% A/50% B
13–13.1 min: 50% A/50% B

Gradient

13.1–14 min: 50% A/50% B to 100% B
14–16 min: 100% B
16–16.95 min: 100% B to 15% A/85% B
16.95–25 min: 15% A/85% B

Flow Rate

1 mL/minInjection
VolumeVariable; set at run time

RESULTS AND DISCUSSIONThe GX-271 Oligo Purification System is configured
with both analytical and preparative injection ports,
selection valves to accommodate different size
chromatography columns, and with the ability to vary
the sample load volume. To illustrate the capabilitiesof this system, we will walk through an example
purification. Crude synthetic oligonucleotides(8–40 mers) were first injected onto a preparative
reverse phase or ion exchange HPLC column andpeaks were collected via conditional fraction
collection. That is, fractions were automaticallycollected by the instrumentation when the Abs260
slope met the criteria established in the method. Thisis shown in Figure 3.

…TRILUTION® LC software facilitated

The instrumentation then removed small samples
from the tubes that contained fractions and injected
them directly onto a reverse phase or anion exchange
analytical column to confirm oligonucleotide sizeand purity (Figure 4 on page 4). In the final
purification step, fractions that passed the previous
size and purity test were automatically injected ontoa size-exclusion desalting column, achieving >90%
oligonucleotide purity. Effective salt removal wasconfirmed by in-line UV and conductivity monitoring.
Modular and flexible programming in theTRILUTION LC software facilitated seamlessexchange of preparative and analytic methods
without manual intervention. On-board fraction
analysis during runs enabled the loading of sample
lists for subsequent runs, resulting in no downtime on
the instrument. Additionally, built-in error handlingconditions at various stages of the process prevented
sample loss and allowed for fully automated,
unattended operation. In the event of an error, therun can be stopped entirely or another method canbe executed to enable sample recovery.
The versatility of the GX-271 Oligo Purification System
permitted continuous sample injection, collection,reinjection, fraction pooling, and desalting on
an automated platform, providing bulk purified
oligonucleotides as the end product for use inmolecular biology applications.
The GX-271 Oligo Purification System makes useof the VERITY® 1810 Conductivity and pH Monitor
to observe salt concentrations. This state-ofthe-art instrument is capable of monitoring salt
concentrations over a large dynamic range with arapid response time in biological purifications, andis also equipped with pH monitoring capabilities.
Figure 5 on page 4 shows an example ofphosphate buffer gradient data collected with theVERITY 1810 Conductivity and pH Monitor.

seamless exchange of preparativeand analytic methods without manualintervention

Figure 3
Pilot Scale Purification of 20 mer Oligonucleotide.

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Figure 4

Analytic Verification of 20 mer Oligonucleotide

Figure 5

Phosphate Buffer Gradient Monitoring with the VERITY® 1810 Conductivity and pH Monitor

REFERENCES
1.

Bonilla, Jose V., and G. Susan Srivasta, eds.
Handbook of Analysis of Oligonucleotides andRelated Products. CRC Press, 2011.

ACKNOWLEDGMENTS
The oligonucleotide purification work wascarried out by Luminex; Madison, WI. TheVERITY® 1810 Conductivity and pH Monitor work wascarried out by Gilson, Inc.; Middleton, WI.
CONCLUSIONSThe challenge of purifying a range ofoligonucleotides (8–40 mers) in various injection
volumes (up to 1.5L) on different chromatography
columns is overcome by the modularity of theGilson instrumentation and the flexibility of theTRILUTION® LC software.

The versatility of the GX-271 Oligo Purification
System and TRILUTION LC software permit easyswitching between hardware components andcolumns accommodating sequential preparative
HPLC, analytical HPLC, and large-scale desalting
functionalities on one system.
On-board fraction analysis during runs enables
on‑the-fly sample addition resulting in no down-timeon the instrument.
Built-in error handling conditions at various stagesof the process prevent sample loss and assure fullwalk‑away capabilities.
Continuous sample injection, collection, reinjection,
fraction pooling, and desalting on a single automatedplatform improves manufacturing efficienciesof oligonucleotides used in molecular biology
applications.
Trademarks
All product and company names are trademarks™ or registered®
trademarks of their respective holders. Use of the trademark(s) in thisdocument does not imply any affiliation with or endorsements by thetrademark holder(s).
MultiCode® is a registered trademark of Luminex.

Gilson, Inc.
3000 Parmenter Street • PO Box 620027 • Middleton, WI 53562 USA
Tel: 608-836-1551 or 800-445-7661 • F 608-831-4451
Gilson S.A.S.
19, avenue des Entrepreneurs BP 145, F-95400 Villiers-le-Bel, France
T +33 (0) 1 34 29 50 00 · F +33 (0) 1 34 29 50 20

www.gilson.com/ContactUs
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