1. High Throughput Oligonucleotide Analysis Using the CombiSep Oligo PRO™ System

2. Introduction
Massive quantities of ssDNA and ssRNA oligonucleotides are produced daily to support the growth of genomic-related applications
These DNA products need to be characterized to ensure proper sizing and acceptable batch-to-batch purity
Traditional slab gel-based methods for characterizing DNA products are time-consuming, labor intensive, not readily automated, and provide relatively poor resolution
While useful for assessing compound identity, Mass Spectrometry cannot quantitatively assess oligonucleotide purity due to size dependent variations in ionization efficiencies
As a result, a bottleneck exists for characterizing the purity of ssDNA oligonucleotides

3. Capillary Gel Electrophoresis (CGE)- + UV
Size-based separation of species possessing a constant mass-to-charge ratio (e.g., denatured SDS-protein complexes, ssDNA oligonucleotides, dsDNA)
Separation medium is a gel-based sieving matrix; smaller sized species migrate faster
Throughput for single capillary CGE methods range from 15 min – 100 min per sample; not adequate for high throughput quality control

4. Principles of MCE-UV Operation
UV light passing through the detection window of a 96-capillary array is imaged onto a linear photodiode array detector
Capillary inlets are arranged 8 x 12 for direct injection from 96-well sample plates
Capillary outlets are bundled to a common reservoir enabling pressure or vacuum to be applied to the array
Samples are separated by the application of a high voltage with optional vacuum flow
96 individual CE separations are performed in parallel with simultaneous UV detection

5. ssDNA Oligonucleotide Analysis by Multiplexed CGE-UV
CGE-UV is the method of choice for assessing oligonucleotide purity, providing higher resolution and superior quantification vs. slab gels.
Label-free UV detection provides a low cost, toxic free alternative and improved quantification compared to fluorescent label approaches for ssDNA which may possess size and sequence dependent fluorescent labeling efficiencies
CGE-UV advantages for characterizing oligonucleotide purity include low sample consumption, high resolving power, direct on-line UV detection and automated operation.
A proprietary gel sieving matrix (Oligel) has been developed for multiplexed operation. When analyzed at low mM concentrations, single nucleotide resolution can be obtained from 10mer to 80mer oligonucleotide lengths, allowing identification of low level n-1, n-2, etc. impurities.
Multiplexed CGE-UV provides a nearly 50-fold improvement in sample throughput over single capillary CE methods with a minimal loss in separation performance. A throughput of 96 samples/h is achieved for 80mer length oligonucleotides.

6. Oligo PRO™ System (CombiSep, Ames, IA USA)
Automatic Tray Handler
Sample Tray #1
Sample Tray #2
Waste Tray
Buffer Tray
Front Access Panel
Second generation 96-capillary array CE instrument
Fixed wavelength UV or visible detection
Slide-out stage accommodates four 96-well plates (1 waste, 1 buffer, 2 samples)
System can be interfaced to a robotic arm for unattended well plate exchange

7. Inside View of the Oligo PRO™ Instrument
Capillary Array Cartridge
Lamp
Housing
HV Power Supply
Syringe
Pump
Optical Platform Housing
Capillary Array Detection Window

8. Capillary Conditioning Solution
High Pressure Pump Option for Oligonucleotide Analysis on the Oligo PRO™ Instrument
Oligel Matrix
Capillary Conditioning Solution
A/B Switching Valve
Pressures up to 400 psi can be applied to the capillary array

9. 96-Capillary Array Viewed from Detector Position
Capillary Outlets
(12 Bundles of 8 Capillaries)
Capillary Inlets
(Arranged in 8 x 12 Format)
Detection Window
(Polyimide coating removed)

10. Capillary Array Inlets Viewed from Below
Direct injection by voltage from 96-well plates
Working injection volume typically 100 ml

11. Image of 96-Capillary Array on PDA Detector
Continuous measurement of UV intensity simultaneously in all 96 capillaries
Absolute light intensity does not have to be equal as the relative absorbance is measured in each capillary

12. Oligo PRO™ Specifications for Oligonucleotide Analysis
Detection
Fixed wavelength UV at 254 nm using mercury lamp and narrow band pass filter
Capillary Array Dimensions
75 mm i.d., 200 mm o.d.
Effective lengths 40 cm to 55 cm; ~20 cm fixed length from detector to outlet
Sample Preparation
Typical oligonucleotide working concentration of 1-5 mM for a standard EK injection
Sample Injection
Electrokinetic; 100 mL working injection volume; -2 to -5 kV for sec
Multiplexed CGE-UV Operational Conditions
Typical total operating current 0.4 – 0.6 mA (<6 mA/capillary)
Field strength ~150 V/cm
Forced air capillary cooling at room temperature

13. Features of Oligel™ Separation Matrix for Multiplexed CE-UV
Contains proprietary mix of polymers and 8 M urea to fully denature oligonucleotides
Low UV background at 254 nm
Low current generation (< 5 mA per capillary at 150 V/cm)
Self-coating of capillaries to reduce EOF
Low viscosity to facilitate faster pumping speeds
Single base resolution from 14 – 80mers
Relatively fast separation speed (80mers in ~1 h)

14. Separation Resolution of Oligel Sieving Matrix
14,15mer
19,20mer
29,30mer
39,40mer
59,60mer
79,80mer
Sample: Desalted 14, ,20 – 29,30 – 39,40 – 59,60 – 79-80mers
Concentration: 1.5 µM (14-30mers); 0.1 µM (39-60mers); 0.2 µM (79,80mers)
CGE-UV: E = -170 V/cm; Sample injection = -5 kV, 10 s; UV = 254 nm

15. 96-Capillary Separation of 14-80mers Using Oligel™ Matrix

16. Analysis of Unmodified (Top) and Amine-modified (Bottom) 70mers
Sample:
Desalted Unmodified, Amine-modified 70mers
Concentration: 5 µM
Injection: -3 kV, 15 s
CE Run: -140 V/cm, 70 min

17. Separation of Various Ratios of Unmodified (A) to
Amine-modified (B) 70mers
A:B=1:9
Sample:
Desalted Unmodified, Amine-modified 70mers
Concentration: 5 µM
Injection: -3 kV, 15 s
CE Run: -140 V/cm, 70 min
A:B=1:3
A:B=1:1

18. Multiplexed CE-UV Analysis of Amine-modified 70mers
Sample: Desalted, Amine-labeled 70mers
Concentration: 8 µM
Sample Injection: -3 kV, 10 sec
CE Run: V/cm, 70 min

19. Characteristics of Gel Matrices Used in CGE
Chemical Gels
Cross-linked, chemically linked to the capillary wall: not replaceable
Well-defined pore structure but pore size cannot be varied after polymerization
Heat sensitive
Particulates can damage gel matrix
Physical Gels
Not cross-linked, not attached to the capillary wall: replaceable
Entangled polymer networks of linear or branched hydrophilic polymers
Dynamic pore structure
Heat insensitive

20. Permanent coating vs. Dynamic coating
Why: for non-cross-linked gel, EOF reduces separation efficiency
How to reduce the EOF?
Permanent coating:
Chemical bonding to the capillary wall
Non-replaceable
Limited life time
Dynamic coating:
Physical bonding (hydrophobicity and charge)
Replaceable

21. Capillary Conditioning Solution
High Pressure Pump Option for Oligonucleotide Analysis on the Oligo PRO™ Instrument
Oligel Matrix
Capillary Conditioning Solution
A/B Switching Valve
Pressures up to 400 psi can be applied to the capillary array

22. Operation Flow Chart (~1 hour) Fill Gel Separation Separation
(400 psi)
(3 to 4 hours)
Total of ten separations
Once a week: Conditional Solution + Gel Fill (~4 hours)

23. Oligonucleotide Analysis : Reagent Cost per SampleDN
Oligel ssDNA Gel, 250 ml
List Price: € 280
Volume Consumed
20 ml/day + 10 ml/run
Plates per Day 6
Samples per Day
576
Cost per sample (Est.)
€0.15 10
960
€0.14 DN
ssDNA Oligel Buffer, 1000 mL
List Price:€102
Volume per Day (ml) - up to 10 runs
106
Samples per Day (6 plates)
€0.02
Samples per Day (10 plates)
€0.01 DN
Capillary Conditioning Solution, 1000 mL
List Price: €153
Volume (ml) per week (Est.)
250
Total Reagent Cost per Sample (6 plates/576 samples/day)
€0.18
Total Reagent Cost per Sample (10 plates/960 samples/day)
€0.16

24. Oligonucleotide Analysis : Capillary Cartridge Cost per Sample
Samples per day
Plates per day
Capillary Lifetime (weeks) 12 16 20 25 36 52 96 1
1,08 €
0,81 €
0,65 €
0,52 €
0,36 €
0,25 €
192 2
0,54 €
0,40 €
0,32 €
0,26 €
0,18 €
0,12 €
288 3
0,27 €
0,22 €
0,17 €
0,08 €
384 4
0,20 €
0,16 €
0,13 €
0,09 €
0,06 €
480 5
0,10 €
0,07 €
0,05 €
576 6
0,11 €
0,04 €
672 7
0,15 €
768 8
0,03 €
864 9
960 10
0,02 €
1248 13
1920
0,01 €
Calculation assumes :
cost per capillary array = €6194
Operating days per week = 5 days/wk

25. Other Technologies Slab Gel Single column capillary electrophoresis
Mass Spectrometers
HPLC

26. Slab Gel Multiple samples analysis Good resolution
Very labor intensive
Difficult to document the image
Semi-quantitation
Long separation time
Acrylamide

27. Single Column Capillary Electrophoresis
Similar resolution as in Oligo PRO™
Better reproducibility
Slow (~96 times slower)
Sample cost: higher due to the use of coated capillary (limited life time)

28. Mass Spectrometers MALDI (matrix assisted laser desorption ionization)
Size determination up to 60’mers
Difficult for quantification
Electrospray ionization
Size determination up to 120’mers
Relatively slow: a couple minutes per sample
HPLC-MS
Size determination & quantification
Slow: half hour to an hour per sample

29. HPLC (Ion Exchanged Chromatography)
Quantitation measurement
Separation up to 40’mers
Slow: half hour per sample
Separation not completely based on size
Better reproducibility

30. Impurity Screening of an Amine-modified 70mer Sample
Approximate % Purity of main peak is 39.1%

31. Co-Winner Best Poster at Tides 2005
CombiSep and IDT Poster Co-Winners of Best Poster at Tides 2005 Conference
The poster titled “Comparisons between Multiplexed, Absorbance-Based Capillary Electrophoresis, Capillary Electrophoresis, and Ion Exchange Chromatography for Analysis of n-1 Oligonucleotide Impurities” by Wei Wei, Ho-ming Pang, Dennis Tallman, and Jeremy Kenseth of CombiSep, Inc and Lisa Bogh of Integrated DNA Technology was recently selected as the co-winner of the best poster at Tides The Tides Conference is an industry event for manufacturing and development of oligonucleotide and peptide products. The meeting was held May 1st – 5th, 2005 at the Boston Convention & Exhibition Center.
The poster award was sponsored by BioProcess International. The selection criteria was based on novelty, applicability, and clarity of data presented.

32. 50:50 Mixture of 29mer:30mer by multiplexed CE-UV, CE-UV, and IEC
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
25 min = 1 Sample
25 min = 1 Sample
IEC method was unable to provide resolution of n-1 species

33. 50:50 Mixture of 39mer:40mer by multiplexed CE-UV, CE-UV, and IEC.
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
27 min = 1 Sample
26 min = 1 Sample
IEC method was unable to provide resolution of n-1 species

34. 50:50 Mixture of 49mer:50mer by multiplexed CE-UV, CE-UV, and IEC.
Multiplexed CGE
70 min = 96 Samples
30 min = 1 Sample
Single Capillary CGE
Ion Exchange HPLC
28 min = 1 Sample
All methods could provide resolution of n-1 species

35. 50:50 Mixture of 59mer:60mer by multiplexed CE-UV, CE-UV, and IEC.
Multiplexed CGE
70 min = 96 Samples
38 min = 1 Sample
Single Capillary CGE
Ion Exchange HPLC
30 min = 1 Sample
IEC method was unable to provide resolution of n-1 species
IEC provided no n-1 resolution at 70mer, 80mer lengths
The CGE methods resolved n-1 at 70mer, 80mer lengths

36. Separation of 36mer and 37mer ssRNA Oligonucleotides
Sample: Two RNA oligonucleotides (36mer, 37mer) were obtained from IDT. The 36mer sequence was 3’-CAGGGACAAGCCCGCCGUGACGAUCUCUAAACAAGC-5’; the 37-mer had an additional A residue on the 3’ end. Samples were diluted to ~ 1 mM in water.
Capillary Array: 75 mm i.d., 150 mm o.d.; 55 cm effective/80 cm total length
CGE: E = V/cm. Sample injection: -2 kV, 15 sec

37. Oligo PROTM Analyzer System
Multiplexed CE-UV system designed and optimized specifically for oligonucleotide analysis and QC
Improved ease-of-use and automation
Enhanced data analysis and report generation capabilities designed with feedback from scientists directly engaged in oligo production
Launched in May 2006

38. Summary
Multiplexed CGE-UV provides a powerful method for oligonucleotide purity analysis, providing superior resolution, throughput and automation compared to slab gel methods
Only minimal sample preparation is required for analysis
The developed Oligel™ matrix is capable of achieving single base resolution from 14-80mers in about 1 h
Multiplexed CGE-UV provides much higher sample throughput and superior resolution to HPLC methods. The standard IEC method could not resolve n-1 species at 30mer, 40mer, or above 60 mer lengths
RNA oligonucleotides as well as dsRNA duplexes can be analyzed for purity in addition to DNA oligonucleotides

39. Details of Disruptive Technologies
Contacts
William Amoyal (Sales and Marketing Director)
Franck Do Vale (Technical Director)
Disruptive Technologies
3 Allée des Camélias
94440 Villecresnes, France
Mobile: +33 (0)
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