Analysis of biomolecules by SEC and Ion-Exchange UPLC

Analysis of biomolecules by SEC and Ion-Exchange UPLC Anders Feldthus, Waters Nordic 2011 Waters Corporation 1

Waters Commitment To develop, commercialize and market columns that when used on Waters ACQUITY UPLC systems, give the speed, sensitivity, resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC. 2011 Waters Corporation 2

Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 3

Ion-Exchange Chromatography Cl - Na + Cl - Na + Cl - Na + Na + Cl - Anion- Exchange Particle Anion- Exchange Particle Cl - Na + Cl - Na + Cl - Cl - Cl - Na + Cl - Na + Cl - Na + Elute with Step or Continuous Binds at Low Ionic Strength Gradients of Increasing Ionic Strength Separations are based on net surface charge on protein with oppositely charged groups on ion-exchanger Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing ph (less common) 2011 Waters Corporation 4

Ion-Exchange Gradient: Starting gpoint Sample High Equilibration Injection Gradient Salt Re-Equilibration Volume Wash unbound proteins ~ 5 cv 10-20 cv 5-10 cv 5-10 cv Typical linear gradient for ion-exchange chromatography 2011 Waters Corporation 5

Ion-Exchange Advantages Moderate resolution Concentrating technique Limitations Separation by charge and charge distribution; insensitive to other properties Can load large volumes of a dilute sample For salt gradients, timeconsuming ph optimization Non-denaturing protein elution techniques Preferred first step when a cost-effective affinity purification is not available required for best resolution. Fractions may need to be desalted prior to the next purification or characterization step (e.g., Mass Spectrometry) 2011 Waters Corporation 6

Protein Isoelectric Points and IEX Particle Support Particle Support 2011 Waters Corporation 7

Strong versus Weak Ion Exchangers Strong and weak DO NOT indicate how tightly the protein is bound A strong exchanger is always ionized i while a weak exchanger s ionization varies with ph Weak Cation Exchange column- pk a 3.5-4.5 Weak Anion Exchange column pk a ~9 Strong exchangers are recommended where there is a need to run at extreme ph (wider operating range) The ion exchange capacity of a weak ion exchanger varies with ph Sample loading (binding) capacity can vary with ph due to loss of charge from the exchanger Selectivity differences exist between weak and strong exchangers Binding constants are a function of the IEX functional group 2011 Waters Corporation 8

Protein-Pak Hi Res IEX 2011 Waters Corporation 10

Attributes of Protein-Pak Hi Res IEX Columns Multi-layered network of ion-exchange groups (SP, CM or Q) o Effective diffusion and binding o High sample loading and resolution o Minimal non-desired interactions No MW limitations: non-porous material QC tested with protein samples for batch-to-batch reproducibility High chemical stability: hydrophilic, polymer-based IEX particles Wide ph range (3-10) high salt concentrations (1M) Standard d pressures (up to 1450 psi for CEX and 2175 psi for AEX) Can be cleaned with aggressive washing ecord enabled for data tracking 2011 Waters Corporation 11

Batch to Batch Reproducibility: Protein-Pak Hi Res Q Column 0.035 0.030 1 2 3 Retention Time Std Dev %RSD 1 - Myoglobin 0.06 1.28 2 - apo-transferrrin 003 0.03 053 0.53 3 - Trypsin Inhibitor 0.03 0.27 0.025 0.020 Batch A 0.015 0.010 Batch B 0.005005 Batch C 0.000 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Reproducible retention time observed for multiple batches 2011 Waters Corporation 12

Carryover 0.16 0.14 1 st Repeat Gradient 2 nd Repeat Gradient 0.12 0.10 0.08 006 0.06 0.04 0.02 Expected Elution Points 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00 No detectable carryover of lysozyme (100 µg injection) 2011 Waters Corporation 13

Ion-Exchange Analysis of Antibodies on Weak Cation Exchange Column 0.040 0.030 0.020 0.010 Humanized IgG1 0.000 0.020 00 0 0.010 Humanized IgG2 0.000 0.015 0.010 0.005 Chimeric IgG1 0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Column: Protein-Pak Hi Res CM, 4.6 x 100mm 2011 Waters Corporation 14

Confirmation of c-terminal Lysine Variants of mab Untreated mab 0.004 K 0.003 KK 0.002 0.001 0.000 0.008 0.006 mab treated with carboxypeptidase B, 30 C, 20 minutes 0.004 0.002 0.000 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00 IEX can be used to confirm the presence of mab lysine variants Column: Protein-Pak Hi Res CM, 4.6 x 100mm 2011 Waters Corporation 15

Strategies to Developing an Ion-Exchange Protein Separation Retention is optimized by adjustment of ionic strength Selectivity is most conveniently optimized with ph Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2011 Waters Corporation 17

Affect of Salt Gradient Slope % 48.00 36.00 24.00 12.00 1 2 Elutes in high salt wash step 0-0.15M NaCl 3 5 Ovalbumin 1 Myoglobin 2 4 Ribonuclease A 3 0.00 28.00 21.00 1 2 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 0-0.3M NaCl 4 5 Cytochrome C 4 Lysozyme 5 % 14.00 7.00 3 0.00 Unbound proteins 48.00 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 1 2 4 5 36.00 % 24.00 3 0-0.5M NaCl 12.00 0.00 0.00 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 Higher salt gradients result in earlier elution of bound proteins High salt wash may be needed in shallower gradients to elute tightly bound proteins Column: Protein-Pak Hi Res CM 4.6 mm x 100mm 2011 Waters Corporation 18

Affect of Salt Gradient Slope: Ovalbumin Variants 8 10 cv gradient 0.02 1 2 3 4 5 6 7 9 10 0.00 6 5 cv gradient 0.02 3 4 1 2 5 7 0.00 6 0.02 3 4 5 7 3 cv gradient 1 2 0.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Longer gradient: increased resolution, lower sensitivity Column: Protein-Pak HI Res Q, 4.6 x 100 mm 2011 Waters Corporation 19

Effect of ph on Selectivity 0.050 0.040 ph 6.6 1 3 α-chymotrypsinogen 1 Ribonuclease A 2 0.030 0.020 2 cytochrome c 3 0.010 0.000 0.050 0.040 0.030 ph 5.0 1 3 0.020 2 0.010 0.000 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 21

Effect of Buffer on Selectivity 0.025 20mM Sodium Phosphate 3 α-chymotrypsinogen 1 1 2 Ribonuclease A 2 0.020 cytochrome c 3 0.015 0.010 0.005 0.000 0.025 20mM MES 0.020 1 Morpholino ethanesulfonic acid 3 2 0.015 0.010010 0.005 0.000 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 Buffer can alter selectivity and retention of proteins at same ph (6) Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 23

Counter Ion Effects 0.020 Ribonuclease A 1 cytochrome c 2 Aprotinin 3 1 2 3 NaCl 0.010 0.000 0.020 KCl 0.010 0.000 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 Counter ion may change selectivity it and retention ti of proteins Effects tend to be minimal 2011 Waters Corporation 24

Temperature Effects 0.005 45 C 0.000 40 C 0.005 0.000 0.005 35 C 0.000 0005 0.005 KK K 30 C 0.000 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Temperature may effect selectivity and retention Changes may be similar to those observed with ph change Column: ProteinP-ak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 26

Waters Auto Blend Plus Technology 12.5% 100mM NaH 2 PO 4 12.5% 100mM Na 2 HPO 4 25mM Sodium Phosphate, 150mM NaCl ph 6.8 15% 1000 mm NaCl 60% H 2 0 2011 Waters Corporation 27

Auto Blend Plus Technology Enter Buffer Concentration Enter Buffer System Enter ph and Salt Gradient 2011 Waters Corporation 28

Effect of ph on Selectivity ph 6 0.020 0.010 Ribonuclease A 1 cytochrome c 2 Aprotinin 3 1 2 3 ph 6.0 0.000 0.020 1 3 2 0.010 ph 6.2 0.000 0.020 0.010 1 3 2 ph 6.4 0.000 1 3 0.020 0.010 2 ph 7.2 0.000 10.00 15.00 20.00 25.00 30.00 35.00 ph can be used to alter selectivity of proteins Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 29

AutoBlend Plus Buffer Management for Protein Ion Exchange Method Development Fluid Path of ACQUITY UPLC H-Class Bio System configured for multi-buffer use The optional solvent select valve provides 6 selectable choices that are associated with particular method names. The proportions of the four selected reservoirs are calculated with Auto Blend Plus Technology to meet the requirements of the method, using the defined buffer system specified with the method. The four physical reservoirs, labeled A, B, C, and D1-D6 can be assigned as needed as the Acid, Base, Salt, and Aqueous components. 2011 Waters Corporation 30

Enhanced Method Development via AutoBlend Plus and Automated Column Management Automated Column Management for Protein Ion Exchange. The method specifies the column to be used and the bypass (B) permits matching the buffer to the column. 2011 Waters Corporation 31

Use of AutoBlend Plus and Automated Column Switching for mab Separation Method Scouting 0.060 0.055 Protein-Pak 0.050 0.045 0.040 0.035 0.030 i k HiRes Q 0.025 0.020 0.015 0.010 0.005 0.000 0.060 0.055 0.050 0.045 0.040 0.035 0.030 0.025 0.020 Protein-Pak HiRes SP 0.015 0.010 0.005 0.000 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 2011 Waters Corporation 32

IEX Summary Total system solution with combination of ACQUITY UPLC H Class Bio System and column Auto Blend Plus Technology allows for change of ph and ionic strengths without preparation of different buffers Simplifies methods development Protein-Pak Hi Res IEX column benefits Consistent batch-to-batch performance (tested with protein standards) Minimal column related carryover Stable over a wide ph range 2011 Waters Corporation 33

Principles of Size Exclusion Chromatography of Proteins Separates proteins by their size in solution (Stokes radius) Separations are Isocratic Tends to be used as a Polishing isolation step or as an analytical technique to determine presence of protein aggregates Generally a lower resolving technique compared to other methods such as ion-exchange or reversed-phase methods 2011 Waters Corporation 35

Size Exclusion Separation of Proteins 2011 Waters Corporation 36

Common Customer Concerns Column-to-column reproducibility Changes in retention time Changes in spacing between peaks Changes in resolution Column lifetime Peak shape deteriorates over time Increased pressure Changes in resolution Tailing of specific proteins Resolution Throughput 2011 Waters Corporation 37

Advantages of UPLC Technology for SEC Separations Requires Columns and Instrumentation to Minimize Band Spreading HPLC Broad Band Broad Peak Less Sensitivity Less Resolving Power Waters UPLC Technology Narrow Peak Increased Sensitivity Increased Resolving Power 2011 Waters Corporation 39

Effect of LC System Dispersion on BEH200 SEC mab Separation Larger system dispersion decreases component resolution 0.30 0.20 USP Res= 1.37 HPLC System BEH200 SEC 1.7um Column (4.6 x 300mm) 0.10 000 0.00 0.25 Waters ACQUITY UPLC System 0.20 USP Res= 2.37 BEH200 SEC 1.7um 0.15 Column (4.6 x 300mm) 0.10 0.05 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 2011 Waters Corporation 40

UPLC-SEC vs HPLC-SEC of mab monomer and aggregates g 0.070 0.070 0.065 0.060 0.055 0.050 ACQUITY BEH200 SEC, 1.7 µm 4.6 x 300mm 0.065 0.060 0.055 0.050 HPLC 100% Silica-Diol SEC 250Å 5µm 7.8 x 300 mm 0.045 0.045 0.040 0.040 0.035 0.035 0.030 0.030 0.025 0.020 0.015 2.26 % Aggregate 0.025 0.020 0.015 2.24 % Aggregate 0.010 0.010 0.005 0.005 0.000 0.000 2.00 4.00 6.00 10.00 8.00 5.00 10.00 15.00 20.00 25.00 30.00 30.00 2011 Waters Corporation 41

ACQUITY BEH200 SEC 1.7 µm Columns Application Areas Determination of protein molecular weight Molecular weight range of 10,000 to 450,000 Daltons Determination of size heterogeneity in a protein sample Quantitation of protein aggregates primarily in therapeutic monoclonal antibodies. 2011 Waters Corporation 42

BEH Overview The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which h provide a stable chemistry with minimal secondary interactions. 2011 Waters Corporation 43

BEH200 SEC, 1.7um Column Batch Test Analyte pi MW 0.22 020 0.20 0.18 0.16 2 4 1. Thyroglobulin, 3 mg/ml 4.6 669,000 2. IgG, 2 mg/ml (Vicam) 6.7 150,000 3. BSA, 5 mg/ml 4.6 66,400 4. Myoglobin, 2 mg/ml 6.8, 7.2 17,000 5. Uracil, 0.1 mg/ml N/A 112 0.14 3 0.12 5 0.10 0.08 0.06 1 0.04 0.02 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 2011 Waters Corporation 44

BEH200 SEC, 1.7um Calibration Curve MW 10000000 1000000 100000 10000 1000 IgG (~150,000 Da) Analyte MW Blue Dextran 2,000,000 Thyroglobulin 669,000 β-amylase 200,000000 IgG, 150,000 Amyloglucosidase 97000 Conalbumin 75000 BSA 66,400 Ovalbumin 44,000 Carbonic Anhydrase 29,000 Myoglobin 17,000 Lysozyme 14,400 Ribonuclease A 13,700 Aprotinin 6,500 Uracil 112 100 10 1 1.5 2 2.5 3 3.5 4 4.5 5 Elution Volume 2011 Waters Corporation 45

Protein Adsorption and Size-Exclusion Chromatography Proteins can interact or adsorb onto the SEC packing material These interactions create undesired and unpredictable retention of proteins (i.e. proteins not separated by size in solution) SEC particles frequently coated with a hydrophilic reagent to minimize non-desired ionic interactions between proteins and packing material Mobile phase additives (e.g., 150mM NaCl) may decrease nondesired ionic interactions between proteins and packing material 2011 Waters Corporation 46

DIOL coating used to minimize non-desired ionic interactions between proteins and packing material Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins SEC Particle - 2011 Waters Corporation 47

DIOL Loss increases non-desired ionic interactions between proteins and packing material SEC Particle - 2011 Waters Corporation 48

Comparative SEC Column Life 0.70 Lysozyme, 0.60 pki = 10.7 HPLC 100% Silica-Diol 0.50 SEC 250Å 4µm 4.6 x 300 mm 0.40 Suggestive of DIOL Bleed 0.30 020 0.20 0.10 Injection 19 Injection 618 0.00 0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 ACQUITY BEH200 SEC, 1.7 µm 4.6 x 150 mm Injection 19 Injection 618 000 0.00 050 0.50 100 1.00 150 1.50 200 2.00 250 2.50 300 3.00 350 3.50 400 4.00 450 4.50 500 5.00 550 5.50 600 6.00 650 6.50 700 7.00 750 7.50 800 8.00 850 8.50 900 9.00 950 9.50 10.0000 BEH200 shows minimal secondary interactions even after 600 injections 2011 Waters Corporation 49

NaCl will decrease ionic interactions between proteins and packing material Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins Na + Cl - Cl - Na + Cl - Na + Cl - SEC Particle Na + Na + Na + Na + Cl - - Na + Na + Cl - Na + Cl - Cl - Cl - Cl - Na + 2011 Waters Corporation 50

Influence of Ionic Strength on Peak Shape and Retention on Silica-based SEC 0.08 006 0.06 0.04 0.02 Conventional 100% Silica-Diol Coated SEC Column 4.6 x 300 mm Lysozyme, pki = 10.7 10mM NaCl 0.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 0.08 0.06 0.04 0.02 lysozyme 25mM NaCl 0.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 008 0.08 0.06 0.04 0.02 lysozyme 100mM NaCl 000 0.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph 6.8 2011 Waters Corporation 51

Influence of Ionic Strength on Peak Shape and Retention on BEH200 SEC 0.24 ACQUITY BEH200 SEC 1.7 µm Column, 0.18 012 0.12 4.6 x 150mm Lysozyme, 10mM NaCl 0.06 pki = 10.7 0.00 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 0.24 0.18 0.12 25mM NaCl 0.06 0.00 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 0.24 0.18 100mM 0.12 NaCl 0.06 0.00 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph 6.8 2011 Waters Corporation 52

Factors Influencing Resolution Resolution decreases with increasing volume and mass loads Resolution decreases with increasing flow rate Ideal flow rate is lower than typically running, however run time will be longer Resolution increases with column length Baseline resolution typically achieved at 50%-100% molecular weight difference Proteins with less than 100% molecular weight difference may not be acceptably resolved 2011 Waters Corporation 54

Mass Loading Capacity on BEH200 SEC, 1.7um 4.6 x 150mm 3.125 µg Total Load Rs = 1.7 400 µg Total Load Rs = 1.5 BSA 0.012 0.012 0.70 0.70 0.010 060 0.60 BSA 0.008 0.50 0.006 BSA dimer 0.40 0.30 BSA dimer 0.004 0.20 0.002 0.10 0.000 0.00 1.00 2.00 3.00 4.00 5.00 6.00 1.00 2.00 3.00 4.00 5.00 6.00 Constant injection volume, 20 µl Resolution decreases for mass loads of 3-400 µg 2011 Waters Corporation 55

Volume Load Capacity on BEH200 SEC, 1.7um 4.6 x 150mm 1.60 1.20 1.00 Injection Volume 1.40 5 1.70 0.80 10 1.54 15 1.43 20 1.27 USP Resolution (Monomer-Dimer) 20 µl 0.60 0.40 5 µl 0.20 000 0.00 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 Constant BSA concentration, 20 mg/ml Resolution decreases for higher injection volume Poor peak shape observed at higher volumes 2011 Waters Corporation 56

Effect of Column Length on mab Rs 20.1% 0.015 BEH200, SEC, 1.7um Aggregate 4.6 x 150 mm 0.010 0.005 USP Res= 1.44 0.000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 0.020 0.015 19.9% 9% Aggregate BEH200, SEC, 1.7um 4.6 x 300 mm 0.010 USP Res= 1.72 0.005005 0.000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Comparable aggregate quantitation Human IgG (Sigma), (load: 10 µg - 150mm; 20 µg -300mm): TUV: 280 nm 2011 Waters Corporation 57

Effect of Flow Rate on mab Rs 0.030 0.020 0.010 0.4 ml/min BEH200, SEC, 1.7um 4.6 x 150 mm 0.000 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 0.030 0.030 0.020 0.020 0.010 0.35 ml/min 0.010 0.000 0.000 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 0.030 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 0020 0.020 0.010 0.2 ml/min 0.000 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Triplicate injections No observable trend in aggregation with flow rate 2011 Waters Corporation 58

Effect of Temperature on mab SEC Peak Shape 0.77 0.70 40 C 45 C BEH200, SEC, 1.7um 4.6 x 150 mm 0.63 0.56 0.49 30 C 0.42 0.35 0.28 0.21 0.14 0.07 0.00 2.10 2.45 2.80 3.15 3.50 Chimeric IgG 0.4 ml/min, 25 mm Sodium Phosphate, 0.15m NaCl, ph 6.8 2011 Waters Corporation 59

Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shape Different cations of chloride salt additive Buffer: 10mM sodium phosphate, ph 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil 2011 Waters Corporation 60

BEH200 SEC, 1.7um Care and Use: (Ways to extend column life) Preparation of SEC Mobile Phase and Needle Wash Pre filter through 0.2 or 0.45 um filter (i.e, Don t inject particulates) Use high purity water Replace mobile phases weekly and do not top off Use of BEH200 SEC, 1.7um Guard Column Attention to SEC Eluent Sinkers Use titanium sinkers NOT stainless steel Sinkers can be major source of bacterial contamination o Consider occasional sinker replacement or 70% alcohol pull through to prevent problems Column Storage Considerations - Overnight: Continuously flush with the mobile phase at 10-20% of the maximum recommended flow rate - Extended: Store in the HPLC grade water with 20% methanol 2011 Waters Corporation 62

BEH200 SEC, 1.7um Guard E(4.6 x 30mm) Extends UPLC SEC Column Life 2011 Waters Corporation 63

QC Protein Standards Mix on Bacterial Contaminated, BEH200 SEC Column BEH200, SEC, 1.7um 4.6 x 150 mm 2011 Waters Corporation 64

Summary: Waters ACQUITY UPLC SEC System Solution New SEC column chemistry based on BEH particles True UPLC separation Application benefits Reduced secondary interaction Improved physical and chemical column lifetime Improved column-to-column reproducibility Improved resolution Improved throughput Synergistic combination of UPLC system and column Higher throughput compared to traditional HPLC 2011 Waters Corporation 65