High Throughput Oligonucleotide Analysis Using the CombiSep cePRO 9600 System™

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

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

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

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  M 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. ssDNA Oligonucleotide Analysis by Multiplexed CGE-UV

cePRO 9600™ System (CombiSep, Ames, IA USA) 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 Automatic Tray Handler Sample Tray #1 Sample Tray #2 Waste Tray Buffer Tray Front Access Panel

Capillary Array Cartridge Lamp Housing HV Power Supply Syringe Pump Optical Platform Housing Capillary Array Detection Window Inside View of the cePRO 9600™ Instrument

High Pressure Pump Option for Oligonucleotide Analysis on the cePRO 9600™ Instrument Oligel Matrix Capillary Conditioning Solution A/B Switching Valve Pressures up to 400 psi can be applied to the capillary array

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

Capillary Array Inlets Viewed from Below Direct injection by voltage from 96-well plates Working injection volume typically 100  l

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

cePRO 9600  Specifications for Oligonucleotide Analysis Detection Fixed wavelength UV at 254 nm using mercury lamp and narrow band pass filter Capillary Array Dimensions 75  m i.d., 200  m 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  M for a standard EK injection Sample Injection Electrokinetic; 100  L working injection volume; -2 to -5 kV for sec Multiplexed CGE-UV Operational Conditions Typical total operating current 0.4 – 0.6 mA (<6  A/capillary) Field strength ~150 V/cm Forced air capillary cooling at room temperature

Contains proprietary mix of polymers and 8 M urea to fully denature oligonucleotides Low UV background at 254 nm Low current generation (< 5  A 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) Features of Oligel™ Separation Matrix for Multiplexed CE-UV

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 14,15mer 19,20mer 29,30mer 39,40mer 59,60mer 79,80mer Separation Resolution of Oligel  Sieving Matrix

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

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

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

Sample: Desalted, Amine-labeled 70mers Concentration: 8 µM Sample Injection: -3 kV, 10 sec CE Run: -150 V/cm, 70 min Multiplexed CE-UV Analysis of Amine-modified 70mers

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

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. Co-Winner Best Poster at Tides 2005

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

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

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

50:50 Mixture of 59mer:60mer by multiplexed CE-UV, CE-UV, and IEC. 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 Multiplexed CGE Single Capillary CGE Ion Exchange HPLC 70 min = 96 Samples 38 min = 1 Sample 30 min = 1 Sample

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  M in water. Capillary Array: 75  m i.d., 150  m o.d.; 55 cm effective/80 cm total length CGE: E = V/cm. Sample injection: -2 kV, 15 sec

Coming Soon: Oligo Analyzer PRO System Multiplexed CE-UV system designed and optimized specifically for oligonucleotide analysis Improved ease-of-use and automation Enhanced data analysis and report generation capabilities designed with feedback from scientists directly engaged in oligo production Scheduled for release in Q2 of 2006

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