Protein PRO Parallel Capillary Electrophoresis System Overview

1. Protein PRO Parallel Capillary Electrophoresis System Overview Advanced Analytical Technologies, Inc. Ames, IA USA

2. Outline • Overview of Protein PRO System • Protein Sizing and IgG Purity Analysis • Parallel Capillary Zone Electrophoresis (CZE) on the Protein PRO • Summary

3. Protein PRO System • 24-Capillary protein electrophoresis separation and quantification system • Superior resolution, quantification and automation vs. traditional slab gel methods • Direct on-line UV absorbance detection of protein with no labeling required • Proprietary capillary conditioning solution paired with gel sieving matrix • Pressure of up to 300 psi can be applied to capillary array • System is open and flexible – can vary capillary i.d., effective length, # of capillaries or separation matrices/methods • Key protein applications: Sizing and Purity by CGE, Charge Heterogeneity by CZE The Protein PRO blends the high resolution and automation of CE with the parallel sampling capabilities of SDS-PAGE

4. Multiplexed (Parallel) CE-UV Technology • 24 capillaries are arranged in a linear array at detection window • UV light is passed through capillary array and imaged onto photodiode array detector • Capillary inlets are arranged 2 x 12 for direct sample injection from 96-well plates or 12-well PCR tube strips • Capillary outlets are bundled to a common reservoir connected to pumping system • Sample injection by vacuum or voltage • 24 individual CE separations are performed in parallel

5. Protein PRO:24-Capillary Array • Capillaries are spread apart throughout entire effective length • Outlet reservoir, ground electrode permanently housed in system

6. Protein PRO:24-Capillary Array Optical Alignment • Continuous measurement of UV light intensity simultaneously in 24 individual capillaries; software automatically assigns and tracks each capillary of array • Absolute light intensity does not have to be equal as the relative absorbance is measured in each capillary

7. Optics and PDA Detector HV Power Supply UV Lamp High Pressure Pump Capillary Array Outlet Reservoir (8 mL) 24-Capillary Array System Configuration

8. Protein PRO: AATI Protein Gel Matrix • Proprietary gel matrix for performing high resolution protein sizing by CE • Uses bare fused silica capillaries and pre-rinse with capillary conditioning solution • Relatively low gel viscosity allows for efficient capillary filling and vacuum sample injection • Low UV background absorbance provides sensitive on-line detection at 214 nm • Relatively low current reduces bubble generation • Gel and conditioning solution can be utilized in single channel CE instruments as well as in Protein PRO parallel CE system • Initial testing with customer demonstrated improved peak shape and reduced tailing for challenging mAb samples as compared to existing single capillary gel and method

9. Capillary Array: 24 capillaries; 75 µm i.d./200 µm o.d.; 33 cm eff/48 cm total New Array Conditioning: 1M NaOH (-2 psi 10 min, hold 20 min), water (300 psi, 5 min), conditioning solution (300 psi, 10 min), protein gel buffer (300 psi, 10 min) Between Run Conditioning: Empty/fill with protein gel @ 300 psi, 10 min CGE Experimental conditions: Pre-run : -4.5 kV 4 min Injection : -0.3 psi 60-120s and hold for 60s (vacuum) or -5 kV 30-60s (ek) Separation : -10 kV to -12 kV Protein PRO: Separation Conditions

10. Protein PRO: Sample Preparation Conditions Non-Reduced CE-SDS: • Prepare 250 mM Iodoacetamide (IAM) alkylating reagent stock solution • Add 10X sample buffer to protein/IgG sample along with IAM and protein markers (optional); load 40 uL solution into wells of 96-well sample plate • Incubate at 70ºC for 10 min; cool to RT for 3 min • Place into Protein PRO instrument for analysis Reduced CE-SDS: • Add 10X sample buffer to protein/IgG sample along with protein markers (optional) and 2-Mercaptoethanol (BME) or similar reducing agent • Load 40 uL solution into wells of 96-well sample plate • Incubate at 70ºC for 10 min; cool to RT for 3 min • Place into Protein PRO instrument for analysis

11. Protein PRO: Protein Sizing Standard Reduced Separation • Run time for reduced 200 kDa protein < 40 min

12. Protein PRO: Protein Sizing Standard Reduced Separation • Raw Data • After normalization to 8.6 kDa and 200 kDa markers

13. Protein PRO: Protein Sizing Specifications Maximum Sample Throughput: Up to 24 different samples in a single CE run Sizing Range: 9 kDa – 200 kDa (other markers may be substituted to adjust sizing range) Dynamic Range: 2-2000 ng/ml Carbonic Anhydrase Sensitivity (S/N > 3): 2 ng/ml Carbonic Anhydrase Sizing Accuracy: Carbonic Anhydrase +/- 3% Sizing Reproducibility: Carbonic Anhydrase < 2% CV Quantitation Reproducibility: CA 14%CV; whole standard series 20%CV (relative concentration to upper marker) Detection Wavelength: 214 nm Run Time: Typical run time 40 min to separate 200 kDa protein Sample Volume Required: Typical 50 mL/sample well (minimum 20 mL/well) Injection Modes: Vacuum or electrokinetic

14. Protein PRO: Lysozyme Calibration Curve 100 ng/ul UB IS 500 ng/ul 200 ng/ul 100 ng/ul 50 ng/ul 20 ng/ul 5 ng/ul 2 ng/ul • Relative to 8.6 kDa ubiquitin internal standard (100 ng/ml) 1 ng/ul

15. L CAII UB IS PB IS 1000 500 200 100 50 20 10 5 2 1 0.5 Protein PRO: α-Lactalbumin and CAII Calibration Curves a-Lactalbumin 2.0 – 2000 ng/mL CAII 2.0 – 2000 ng/mL • Phosphorylase B (97 kDa) used as internal marker • Dynamic range corresponds to 0.1% level detection

16. HC LC IS NGHC Protein PRO: IgG Purity/Heterogeneity • Sample: Beckman IgG Control Standard, Reduced Conditions

17. HC LC NGHC Protein PRO: Reduced IgG Separation • Sample: 1 mg/mL Beckman IgG Control Standard, Reduced Conditions • Non-glycosylated heavy chain can be resolved from heavy IgG chain, allowing for evaluation of % glycosylation • Direct UV detection of protein – exact same method as “gold standard” single capillary method – with > 20X increase in throughput

18. Reduced IgG Separation using Protigel Matrix • Sample: 1 mg/mL Beckman IgG Control Standard, Reduced Conditions • Six consecutive run profile on Beckman MDQ instrument using AATI Protigel CE-SDS separation matrix and method • Kit is available for single capillary CE systems

19. 10 kD Protein PRO: Non-Reduced IgG with 10 kDa Marker Intact IgG (~150 kDa) LC HC • Some light chain/heavy chain as well as other lower MW impurities are observed • Both reduced and non-reduced analysis can be performed in parallel in different capillaries during a single experiment

20. HC LC NGHC 10 kD Customer Reduced IgG Sample with 10 kDa Marker • Result was comparable to customer’s existing single capillary CGE method

21. Intact IgG LC HC 10 kD Customer Non-Reduced IgG Sample with 10 kDa Marker • Result showed slightly improved resolution compared to customer’s existing single capillary CGE method

22. Standard Protein Mixture: 50 um i.d. Capillaries • System can be easily switched from 75 um to 50 um i.d. capillary array to optimize or adjust methods • 50 um i.d. capillaries may further increase separation resolution, improve peak shape

23. Parallel CZE-UV: Small Molecule Mixture Sample: 2 Pyridinium Cations, DMSO, 2 Benzene Sulfonic Acid Anions Capillary Array: 50 um i.d.; 55 cm EFF, 80 cm TOT; 24-capillary BGE: 10 mM Sodium Tetraborate pH 9.2 CZE Conditions: -0.5 psi, 20 sec inj +12 kV (150 V/cm) 214 nm UV detection

24. Normalized Migration Time (Peak 5 vs. Peak 4) Normalized Corrected Peak Area (Peak 5 vs. Peak 4) Parallel CZE: Migration Time and Peak Area Reproducibility • Reproducible, high throughput CZE methods can be developed on same platform used for CGE methods

25. Key Benefits and Summary • Parallel CE-UV technology utilized with the Protein PRO system can be applied to a broad range of high throughput bioanalytical applications • Parallel CE-UV provides many benefits: • Significantly increased sample throughput • Improved laboratory efficiency • Lower turnaround times • Decreased reagent and sample consumption • Reduction in labor and operational/maintenance costs • ONLY technology providing high throughput CGE and CZE capabilities • Many methods previously developed for single capillary CE instruments can be successfully transferred to a parallel format • The parallel CE configuration format provides an open, flexible format to vary capillary length, i.d., # capillaries, internal markers and/or separation conditions to adjust resolution or accommodate different applications as needed

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