Size Exclusion BioHPLC Columns

Size Exclusion BioHPLC Columns Size Exclusion Product Families Particle Porosity Functionalities Particle Pore Size Application Sizes Agilent Bio SEC- Silica Fully porous N/A um 00A, 0A, 00A High efficiency Silica Fully porous N/A um 00A, 0A, 00A More pore sizes 00A, 000A, 000A ProSEC 00S Silica Fully porous N/A um 00A Extend linear range broader sample range ZORBAX GFC Silica Fully porous N/A um, 6um 0A 00A Larger column sizes. High efficiency small particles. Multiple pore sizes to cover broad range of molecular sizes. Single pore size to cover range of globular proteins/antibodies Page 6. Larger columns for fractionation

SEC Applications Non-Denaturing Conditions Separations Based on Size in Solution Impurity testing, aggregation g analysis (separation of monomer/dimer/aggregates) Molecular weight characterization over wide MW range (000 0M) possible Expression and folding studies Separation of Reaction Components and Products, (conjugates) Purification Desalting and salt exchange Collection of fractions under non-denaturing conditions Page 6 SEC Technique Technique is isocratic No interaction with the stationary phase Separation based on differences in size Eluent contains buffer and salt Technique does not denature proteins 0,000 µv 00,000 0,000 00,000 0,000 00,000 0,000 00,000 0,000 00,000 0,000 Decreasing size R&D Bio SEC.DATA Smaller molecules spend longer in the pores and elute later. Larger molecules spend less time in the pores and elute sooner. 0 RT [min] 0 Gel Filtration Standard ( vial diluted with mls of eluent) (Thyroglobulin, -globulin, ovalbumin, myoglobin & vitamin B) 6 7 8 9 0

Elution Profiles If sample molecules have the same molecular dimensions, they will coelute and may not be separated by this technique If two molecules have the same molecular weight but different size in solution they may be separated The calibration curve describes how different size molecules elute fromthecolumnandcanbeusedto determine molecular weight equivalents based on solution size Choose the Appropriate Pore Size Pore Size MW Range Typical Sample (Globular Protein) 00Å 00 00,000 Small proteins 0Å 00 0,000 Small proteins 00Å,000,0,000 Most protein samples 00Å,000,000,000 Larger proteins and antibodies 000Å 0,000 7,00,000 Very large proteins 000Å > 0,000,000 Very large molecules viruses Lower limit on this column is hard to define, Use only for special applications 6

Example of Choice of Pore Size Data. Thyroglobulin Aggregates-. Thyroglobulin-MW 670KD.. -globulin- MW 8 KD. Ovalbumin- MW KD 6. Myoglobin- MW 7KD 7. Vitamin B-MW.KD Eluent: Columns: Flow: Detector: System: 0mM Na HPO, 0mM NaH PO + 0.M NaCl, ph6.8 Agilent Bio SEC- 00Å, 0Å & 00Å µm.6x00mm 0.ml/min UV@0nm Agilent 60 Infinity Bio-Inert LC System. Dimer.. Monomer... Monomer Fragment.. Azide. Retained Molecule Bio-Rad Gel Filtration Standards Mix 6 7 Mouse IgG 00Å 0Å 00Å 0Å 00Å 00Å 7 Separation of Recombinant EPO-Protein (MW: kda) Agilent BioSEC-, 00A.6 x 00mm Column: Agilent Bio SEC, 00A.6 x 00mm ID Sample: repo protein (0.mg/ml) Buffer: 0mM Na phosphate, ph 7.0 Flow rate: 0.ml/min Temp: C Gradient: isocratic Detection: UV Baseline expansion

Monitoring Aggregation and Impurities (IgG MW; 0,000 Da) BioSEC- 00Å, Phosphate buffer to control ph and provide ionic strength Optimized to eliminate interactions 0.0 Column: Bio SEC- 00Å, 7.8x00mm Mobile phase: 0 mm Phosphate, ph 7 0.00 Flow rate:.0 ml/min Temperature: Ambient 0.0 Sample: Monoclonal antibody (0 L, mg/ml) 0.00 0.0 0.00 0.00 MAb Aggregates MAb Dimer Low Molecular Weight Impurities Buffer/Excipients 0.000-0.00.00.00.00.00.00 6.00 7.00 8.00 9.00 0.00.00.00.00.00.00 utes 9 Match Particle Size to Flow Rate and Column Pressure Pressure Increasing flow rate reduces efficiency and resolution.0ml/min.ml/min.0 ml/min monomer dimer Flow Rate Particle Size Column: Agilent Bio SEC-, 7.8 x0mm Sample: mab (mg/ml) Injection: ul Flow rate:.0,. and ml/min (6 bar, 7 bar, 0 bar) Eluent: 0mM sodium phosphate + 00mM Na-sulfate Detection: 0nm Flow Rate Resolution Monomer/Dimer Monomer Efficiency Percentage Dimer.0ml/min.,0 0.6.ml/min.,0 0. 7 utes.0ml/min.,97 0.6 0

Agilent NEW Size Exclusion Columns Agilent Bio SEC- Unique, m particle 00Å, 0Å, 00Å pore sizes Highest resolution Highest efficiency Faster SEC separations m Particle 00Å, 0Å, 00Å, 00Å, 000Å, 000Å pore sizes High stability and long lifetime Great reproducibility Comparing Agilent Bio SEC- and Bio SEC- Improved Efficiency With Smaller Particles Agilent Bio SEC-, 00Å, 7.8x00mm Column Pressure Bio SEC- (7.8x00mm) 9 bar Bio SEC- (7.8x00mm) bar Peak Protein SEC-, 00Å (7.8x00mm) SEC-, 00Å (7.8x00mm) Thyroglobulin 60 0 BSA Dimer 00 70 BSA 090 690 Ribonuclease A 000 60 Uracil 800 7860, 00Å, 7.8x00mm 0 6 7 8 9 0 Column: Bio SEC- 00Å and Bio SEC- 00Å Buffer: 0 mm Phosphate buffer, ph 7 Flow rate:.0 ml/min for 7.8x00 mm Temperature: Ambient (~ C) Detection: UV nm Injection: 0 µl ( L for.6x00 mm) Sample: ) Thyroglobulin (.0 mg/ml), 670 kd; ) BSA dimer, kd; ) BSA (.0 mg/ml), 66 kd; ) Ribonuclease A (.0 mg/ml),.7 kd, and ) Uracil (. g/ml), 0D. 6

Agilent Bio SEC- Sharp Peaks and High Resolution of Proteins Agilent Bio SEC-, 0Å, 7.8x00mm Agilent Bio SEC-, 00Å, 7.8x00mm 0.0.0.0.0.0.0 6.0 7.0 8.0 9.0 0.0.0.0.0.0.0 Peak Compound SEC- 0Å SEC- 00Å Thyroglobulin 800 60 BSA Dimer 0 0 BSA 60 090 Ribonuclease A 7600 000 Columns: Bio SEC-, 7.8x00 mm Mobile phase: 0 mm Phosphate, ph 7 Flow rate:.0 ml/min Temperature: Ambient (~ C) Detection: UV nm Injection: 0 µl Sample: ) Thyroglobulin (.0 mg/ml), 670 kd ) BSA dimer, kd ) BSA (.0 mg/ml), 66 kd ) Ribonuclease A (.0 mg/ml),.7 kd ) Uracil (. g/ml), 0D Uracil 000 800 Agilent Bio SEC- Bio SEC-, 0Å, Capacity: BSA Loading Test.00 Injection volume: 0 L BSA Concentration.80 (from low to high): mg/ml 60.60 mg/ml mg/ml.0 0 mg/ml.0 0 mg/ml 0 mg/ml.00 0.80 0.60 0.0 00 0.0 Column: Bio SEC-, 0Å, 7.8x00mm Mobile phase: 0 mm Phosphate, ph 7.0 Flow Rate:.0 ml/min Injection volume: 0 µl Detector: nm 0.00 0.0.0.0.0.0.0 6.0 7.0 8.0 9.0 0.0.0.0.0.0.0 7

Agilent Bio SEC- Separations of E. coli lysate Bio SEC- 0Å, 7.8x00mm Bio SEC- 00Å, 7.8x00mm 00 0.0 0.0 0.0 0.0 0.0 0.0 60 6.0 70 7.0 80 8.0 90 9.0 0.00.0.0.0.0.0 Column: Column: Bio SEC- 0Å, 7.8x00mm & Bio SEC- 00Å, 7.8x00mm Mobile phase: 0. M Phosphate, ph 7.0 Flow rate:.0 ml/min Detection: UV nm Temperature: Ambient Injection: 0 µl Sample: E. coli lysate (. mg/ml) Agilent Bio SEC- Monoclonal Antibody Aggregation Monitoring 0.0 Column: Bio SEC- 00Å, 7.8x00mm Mobile phase: 0 mm Phosphate, ph 7 0.00 Flow rate:.0 ml/min Temperature: Ambient 0.0 Sample: Monoclonal antibody (0 L, mg/ml) 0.00 0.0 0.00 0.00 0.000000-0.00.00.00.00.00.00 6.00 7.00 8.00 9.00 0.00.00.00.00.00.00 utes 8

High Capacity and High Resolution Au nm m 00 00 0 0 Å. thyroglobulin,. min. BSA dimer, 6.9 min. BSA monomer, 6.9 min. ribonuclease A, 8.7 min. poly-dl-alanine (- kda), 9.90 min 6. uracil,. min 0 6 8 0 Time (min) nm m 00 0 00 TSK G000SW xl. thyroglobulin,.6 min. BSA dimer, 6. min. BSA monomer, 7.0 min. ribonuclease A, 9. min. poly-dl-alanine (- kda), 0.0 min 6. uracil,.8 min 0 6 8 0 Time (min) High Capacity and High Resolution Columns: Bio SEC-,.6x00 mm Buffer: 0. M Phosphate buffer, ph 7.0 Flow rate: 0. ml/min Detector: UV nm Injection volume: 0 µl.thyroglobulin. -Globulin. Ovalbumin. Ribonuclease A. p-aminobenzoic acid Bio SEC-, 0Å Bio SEC-, 00Å Bio SEC-, 00Å 0.00.00.00 6.00 8.00 0.00.00.00 9

High Molecular Weight Protiens 0. Column: Bio SEC-, 000Å,.6x00mm Buffer:0 mm phosphate buffer, ph 7.0 0.0 Flow rate: 0. ml/min Detection: nm 0.8 Temperature: Ambient 0.6 0. Proteins:.Thyroglobulin (670kD). Uracil (0D) 0. 0.0 0.08 0.06 0.0 Possible Dimer 0.0 0.00 0 6 8 0 Reproducibility, 00Å, 7.8x00mm Buffer: 0 mm Phosphate buffer, ph 7.0 Flow Rate:.0 ml/min Detector: nm Injection: 0 ul.thyroglobulin. BSA. Ribonuclease A. Uracil.0.0.0.0.0 6.0 7.0 8.0 9.0 0.0.0.0.0.0.0 utes 0

Lot to Lot Reproducibility, 00Å, 7.8x00mm 0.0 0.8 0.6 0. 0. 0.0 0.08 Black: SN 086 Lot# 00907MGB Blue: SN 0 Lot# 0606MG Green: SN 7 Lot# 006CL 0.06 0.0 0.0 0.00 0.00.00.00.00.00.00 6.00 7.00 8.00 9.00 0.00.00.00.00.00.00 utes Lot Thyroglobulin BSA Ribonuclease A Uracil RT Eff. RT Eff. RT Eff. RT Eff. 00907MG 6.7 6 9. 678 0. 7.0 87 0606MG 6.7 7 9. 68 0.6 06. 69 006CL 6.77 60 9.9 6 0.7 8. 6709 Stability at ph 8.. Thyroglobulin. BSA. Ribonuclease A. Uracil Bio SEC-, 00Å,.6x00mm Buffer: 0mM Phosphate, ph 8. Wavelength: nm Flow Rate: 0. ml/min Injection: ul Pre-test, ph7.0 Day AM, ph8. Day PM, ph8. Day AM, ph8. Day PM, ph8. Day AM, ph8. Day 7 AM, ph8. 0 6 7 8 9 0 utes

Stability at ph 8., increasing injection volume.00 Column: Bio SEC-, 00Å, 7.8x00mm, ph 8..80 Sample: BSA Concentration: mg/ml 60.60 Mobile Phase: 0 mm Phosphate, ph 8..0 Flow Rate:.0 ml/min Detector:.0.00 0.80 0.60 0.0 0.0 0.00 0.00 0.0.00.0.00.0.00.0.00.0.00.0 6.00 6.0 7.00 7.0 8.00 8.0 9.00 9.0 0.00 0.0.00 utes Injection volumes ul, 0 ul, 0 ul, 0 ul, and 00 ul Monoclonal Antibody Aggregation Monitoring 0.00 0.08 0.06 0.0 0.0 0.00 0.008 0.006 0.00 0.00 0.000-0.00-0.00-0.006-0.008 Columns: Bio SEC-, 00Å, 7.8x00mm Buffer: 0 mm Phosphate, ph 7 Flow Rate:.0 ml/min Sample: Mab Temperature: Ambient MAb Dimer Mab Aggregates Buffer Peak.0.00.0.0.0 6.0 7.0 8.0 9.0 0.0.0.0.0.0.0

Size Exclusion Separations of E. coli Lysate (Single Columns) Columns: Bio SEC-, 7.8x00mm Buffer: 0 mm Phosphate, ph 7.0 Flow Rate:.0 ml/min Detector: nm Injection: 0 µl (. mg/ml) SEC-, 000Å SEC-, 00Å SEC-, 00Å SEC-, 0Å 0 6 7 8 9 0 Size Exclusion Separations of E. coli Lysate (Columns In Series)...0 Columns: Bio SEC-, 000Å, 7.8x00mm + Bio SEC-, 0Å, 7.8x00mm Buffer: 0 mm Phosphate, ph 7.0 Flow Rate:.0 ml/min Detector: t nm Injection: 0 µl (. mg/ml) 0.8 0.6 0. 0 0. 0.0 0 6 8 0 6 8 0 6 8 0 utes