New ProSwift Monolith Reversed-Phase and Ion-Exchange

ISPPP 2007 Presentation 1

ABSTRACTMonolith columns offer significant advantages over conventional packed columns with porous substrates. These advantages include fast mass transfer, high loading capacity, improved resolution at elevated flow rates,lower backpressure, and wide pH stability. These exclusive char-acteristics support versatile performance in a wide range of biomolecule separations. Dionex has introduced reversed-phase anion- and cation-ex-change phases of ProSwift™ monoliths (4.6 × 50 mm). Reversed-phase ProSwift columns (4.6 × 50 mm) include RP-1S, RP-2H and RP-3U ver-sions. Each differs in pore structure and selectivity for various biosepara-tions. The low backpressure of the ProSwift RP columns support fast separation of analytes at high flow rates, providing increased productivity. ProSwift ion-exchange phases (4.6 × 50 mm) include weak anion- ex-change (WAX-1S) and strong anion-exchange (SAX-1S) columns, and a weak cation-exchange (WCX-1S) column. The most recent additions to the monolithic column family line include the 1 × 50 mm WAX-1S and WCX-1S columns, developed especially for high resolution microana-lytical biomolecule separations. The 1 mm columns offer improved sensitivity and reduced solvent consumption. Using both reversed phase and ion-exchange columns, we have developed various applications and compared with competitor columns. These results will be presented.

INTRODUCTIONProSwift monolithic columns, available with either reversed-phase or ion-exchange chemistries, are specifically designed to provide high-resolution, high-efficiency separations of proteins, peptides, and other biomolecules, using conventional HPLC systems.

A monolith consists of aggregations of globules that resemble cauliflow-er in appearance.The open spaces between these aggregates are large flow-through channels, which help to minimize column backpressure. The spaces among the smaller globules are open or “through-pores” which allow the sample to interact with the surface of the media quickly. The mass transfer of the sample is primarily driven by convective flow through these open pores, instead of molecular diffusion, which is much slower. These pores are big enough to allow even large molecules to flow through freely. (Most small molecules are less than 500 nm.)

Therefore, the path lengths for mass transfer through these small globules are much shorter than the path lengths in conventional bead-based chromatographic phases. In addition, the globules are essentially non-porous (based on nitrogen adsorption (BET) measurements and scanning electron microscopy (SEM) examinations). Thus, diffusion-controlled mass transfer is minimized using these columns. By contrast, diffusion controlled mass transfer is a predominant feature of columns packed with porous beads.

Features of ProSwift Monoliths• Highspeedandhighresolution• Fastmasstransfer• Lowbackpressures• Widerangeofoperationalflowrates(RPColumns)• Highthroughputandimprovedproductivity• Highloadingcapacity(IEXColumns)• ExcellentstabilityoverawidepHrange• Outstandingreproducibility• Optimalperformance

MATeRIAlSChromatographic components RP: P680 HPG gradient pump, UVD 340 absorbance detector, UltiMate® 3000 autosampler and TCC-100 Thermostatted Column Compartment from Dionex Corporation.

IEX: ICS-3000 DP gradient pump, VWD Absorbance detector (or, UVD 340), AS autosampler, and TCC-100 Thermostatted Column Compartment from Dionex Corporation.

Chromatography was controlled by Chromeleon® Chromatography Management software (Dionex Corporation).Proteins used in standards, MES, Tris and all other analytical grade chemicals were obtained from Sigma-Aldrich Co.

ISPPP 2007 Presentation

Srinivasa Rao, Kelly Flook, Jim Thayer, Charanjit Saini, Maria Rey, Andy Woodruff, Yury Agroskin and Chris Pohl, Dionex Corporation, Sunnyvale, CA, 94085

New ProSwift Monolith Reversed-Phase and Ion-Exchange Columns and Their Comparative Evaluation with Other Biocolumns

New ProSwift Monolith Reversed-Phase and Ion-Exchange Columns and Their Comparative Evaluation with Other BiocolumnsSrinivasa Rao, Kelly Flook, Jim Thayer, Charanjit Saini, Maria Rey, Andy Woodruff, Yury Agroskin and Chris Pohl, Dionex Corporation, Sunnyvale, CA, 94085

Anuta

New Stamp

2 New ProSwift Monolith Reversed-Phase and Ion-Exchange Columns and Their Comparative Evaluation with Other Biocolumns

Monolithic Columns from Dionex CorporationProSwift RP-1S 4.6 × 50 mm, P/N 064297

ProSwift RP-3U 4.6 × 50 mm. P/N 064298

ProSwift RP-2H 4.6 × 50 mm, P/N 064296

ProSwift SAX-1S 4.6 × 50 mm, PEEK-lined stainless steel, P/N 064293

ProSwift WAX-1S 4.6 × 50 mm, PEEK-lined stainless steel, P/N 064294

ProSwift WAX-1S 1 × 50 mm, PEEK, P/N 066642

ProSwift WCX-1S 4.6 × 50 mm, PEEK-lined stainless steel, P/N 064295

ProSwift WCX-1S 1 × 50 mm, PEEK, P/N 066643

Columns from Other ManufacturersCompetitor A: RP Leading biocolumn

Competitor B: Leading strong anion-exchange column

Competitor C: Leading weak cation-exchange column

ReveRSeD PhASe

Figure 1. The ProSwift family.

Figure 2. Separation of protein standard mixture on RP-2H with increasing flow rates.

Figure 3. Column backpressure vs. flow rate.

23874-01

Product Line

Reversed-Phase Ion-Exchange

4.6 × 50 mm 4.6 × 50 mm 1 × 50 mm1 mm

In DevelopmentProSwift RP-3U

ProSwift RP-2H

ProSwift RP-1S

ProSwift WAX-1S ProSwift WAX-1S

ProSwift WCX-1S

ProSwift SCX-1S

ProSwift SAX-1S

ProSwift WCX-1S

ProSwift SAX-1S

Key: Current Products Current Development

0 1 2 3 4 5 6 7

1

2

34

5

8 9 10 11

mAU

Minutes

8 mL/min4 mL/min

2 mL/min1 mL/min

FlowRate: 1, 2, 4, or 8 mL/minInj. Volume: 5 µLTemperature: 30 °CDetection: UV, 214 nmSample: Mixture of five proteins

Peaks: 1. Ribonuclease A 1.5 mg/mL 2. Cytochrome C 0.5 3. BSA 1.5 4. Carbonic anhydrase 0.9 5.Ovalbumin 1.5

22903

Column: ProSwift RP-2H, 4.6 × 50 mm Eluents: A. DI H2O/CH3CN95:5(v/v)+0.1%TFA B. DI H2O/CH3CN5:95(v/v)+0.1%TFA

Gradient: 1 mL/min: 1-75% B in 12 min 2 mL/min: 1-75% B in 6 min 4 mL/min: 1-75% B in 3 min 8 mL/min: 1-75% B in 1.5 min

00 2 4

Flow Rate (mL/min)6 8 10

500

1000

1500

2000

2500

Colu

mn

Back

pres

sure

(psi)

22905-01

Polymer Beads, 8 µm

ProSwift RP-1S

ProSwift RP-2H

ProSwift RP-3U


Figure 4. Peptide and protein separation on RP columns.

Figure 5. pH stability of the ProSwift RP-3U.

ReveRSeD PhASe— COMPeTITOR COMPARISON

Figure 6. Comparison of RP-2H with competitor A column; separation of protein standard mixture.

Figure 7. Comparison of ProSwift RP-1S with competitor A column; separation of pancreatin.

RP-3U

RP-2H

RP-1S

0 2 4 6 8 10 12–5

250

mAU

Minutes23132

Flow: 1.5 mL/minGradient: 1–64% B in 17 min

Sample: 1. Methionine enkephalin acetate (Tyr-Gly-Gly-Phe-Met) (MW 573.7) 2. Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) (MW 555.6) 3. Angiotensin II acetate (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (MW 1046.2)

4. Physalaemin (Glu-Ala-Asp-Pro- Asn-Lys-Phe-Tyr-Gly-Leu-Met) (MW 1265.4) 5. Substance P acetate (Arg-Pro-Lys- Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) (MW 1347.6) 6. Ribonuclease A 7. Cytochrome C 8. Carbonic anhydrase 9. Bovine serum albumin 30 µg/mL each

No treatment

0.1M HCl48 hrs

1M NaOH48 hrs

Flow: 4 mL/minSample: 1. Ribonuclease A 0.1 mg/mL) 2. Cytochrome C 0.05 3. BSA 0.1 4. Carbonic anhydrase 0.1

325

0.6 0.90.3 1.2

1 23 4

Minutes23137

0–5

mAU

1.5

This figure shows the resolution of four protein standards before and after base and acid regeneration treatments. The ProSwift RP monolith column is stable with treatment of1 M NaOH and 0.1 M HCl in the regeneration process. It is shown to be stable at pH 1 to 14at normal temperature and flow for long periods.

0 3 9–100

700

Minutes

ProSwift RP-2H

ProSwift RP-2H

Competitor A

Inj. Volume: 5 µLTemperature: 30 °CDetection: UV, 214 nmSample: Mixture of five proteins

Peaks: 1. Ribonuclease A 1.5 mg/L 2. Cytochrome C 0.5 3. BSA 1.5 4. Carbonic anhydrase 0.9 5. Ovalbumin 1.5

1 23 4 5

22904

2 mL/min

0 1 20

4508 mL/min

6

Columns: 1. ProSwift RP-2H, 4.6 × 50 mm 2. Competitor A, 4.6 × 100 mm, 15 µmEluents: A. DI H2O / CH3CN, 95:5 (v/v) + 0.1% TFA B. DI H2O / CH3CN, 5:95 (v/v) + 0.1% TFA

Gradient: 2 mL/min: 1–75% B in 6 min 8 mL/min: 1–75% B in 1.5 min

Flow Rate: 2 or 8 mL/min

24492

Minutes

Column : A. ProSwift RP-1S, 4.6 × 50 mm B. Competitor A, 4.6 × 100 mmEluents: A. DI H

2O,CH

3CN 95:5 (v/v)

+0.1%TFA B. DI H

2O,CH

3CN 5:95 (v/v)

+0.1%TFA

0 2 4 6 8 10 12 14 16 18

1,400

mAU

mAU

0

1,400

Flow Rate: 4 mL/min

ProSwift RP 1S

Flow Rate: 1 mL/min

0 1 2 3 4 4.6

ProSwift RP 1S

Competitor A

Gradient: Flow1mL:1–75%Bin12min Flow4mL:1–75%Bin3min

Inj. Volume: 10 µLDetection: UV at 214 nmSample: Pancreatin


Figure 8. Comparison of ProSwift RP-1S with competitor A column; separation of thrombin.

ANION exChANge

Figure 9. Separation of protein mix on WAX-1S.

Figure 10. Separation of E. coli proteins on the WAX-1S.

Figure 11. Protein loading on WAX-1S.

24493

Minutes

Gradient: Flow1mL:1–75%Bin12min Flow4mL:1–75%Bin3min

Inj. Volume: 10 µLDetection: UV at 214 nmSample: Thrombin

0 2 4 6 8 10 12 14 16 18.1

300

-50

mAU

mAU

0

250

Flow Rate: 4 mL/min

ProSwift RP 1S

Flow Rate: 1 mL/min

0 1 2 3 4 4.6

1

2 ProSwift RP 1S

Competitor A

Column : A. ProSwift RP-1S, 4.6 × 50 mm B. Competitor A, 4.6 × 100 mmEluents: A. DI H2O,CH3CN, 95:5 (v/v) +0.1%TFA B. DI H2O,CH3CN, 5:95 (v/v) +0.1%TFA

24269

Minutes

Column: ProSwift WAX-1S, 1 × 50 mm Eluents: A. 10 mM Tris pH 7.6 B. 1 M NaCl in 10 mM Tris, pH 7.6

Gradient: 5 to 55% of B in 13 min, hold for 2 min

FlowRate: 0.2 mL/min

0 2 4 6 8 10 12 14-2

20

mAU 1

2

3

Inj. Volume: 1.3 µLTemperature: 30 °CDetection: UV, 280 nmSample: Protein mixture

Peaks: 1.Ovalbumin 1 mg/mL 2. Trypsin inhibitor 1 3. Insulin chain A 0.25

24270

Minutes

Inj. Volume: 1.3 µLTemperature: 30 °CDetection: UV, 280 nmSample: 1.25 mg/mL E. coli protein

0 5 10 15

mAU

Column: ProSwift WAX-1S, 1 × 50 mmEluents: A. 10 mM Tris, pH 7.6 B. 1 M NaCl in 10 mM Tris, pH 7.6

Gradient: 5 to 55% of B in 13 min, hold for 2 min FlowRate: 0.2 mL/min

24343

0.185

0.168

0.1450.138

0.139

1

2

3

4.5 µg 9.0 µg

36 µg 18 µg

54 µg

PWHH

mAU

Minutes0 12

00 0.6 1.1 1.6

0.1

0.2PW

HH (m

in)

Column: ProSwift WAX-1S, 1 × 50 mmEluents: A. 10 mM Tris, pH 7.6 B. 1M NaCl in eluent A

Gradient: 5–55%Bin7.5min


Log Sample Load (µg)

Inj. Volume: 2–24µLTemperature: 30 °CDetection: UV, 214 nm

Sample: 1.Ovalbumin,1mg/mL 2. Trypsin Inhibitor, 1 mg/mL 3. Insulin Chain A, 0.25 mg/mL


Figure 13. Comparison of ProSwift SAX and competitor B columns; separation of pancreatin.

ANION exChANge— COMPeTITOR COMPARISON

Figure 12. Comparison of ProSwift SAX and competitor B columns; separation of protein mixture.

CATION exChANge

Figure 14. Separation of monoclonal antibody (MAb) on WCX-1S.

Figure 15. Loading capacity of MAb on WCX-1S.

Column: ProSwift SAX, 4.6 × 50 mm Competitor B, 5 × 50 mmEluents: A. 10 mM Tris, pH 7.6 B. 10 mM Tris + 1M NaCl, pH 7.6

Gradient: 2–50% B in 5 min

Flow Rate: 1.5 mL/min

1200

2 31 4Minutes

23137

0–200

mAU

5 6 7 8 9

Competitor B

ProSwift SAX

1

2

3

4

Inj. Volume: 10 µLDetection: UV, 214 nm

Sample: Mixture of four proteins, 1 mg/mL each 1. Myoglobin 2. Conalbumin 3. Ovalbumin 4. Trypsin inhibitor

2000

2 31 4Minutes

24494

0–200

mAU

5 6 7 8 9

Competitor B

ProSwift SAX1

2

Column: ProSwift SAX, 4.6 × 50 mm Competitor B, 5 × 50 mmEluents: A. 10 mM Tris, pH 7.6 B. 1M NaCl in Eluent AGradient: 2–50% B in 5 min Flow Rate: 1.5 mL/min

Inj. Volume: 10 µLDetection: UV, 214 nmSample: Pancreatin 36 mg/mL + 5 mM DTT resuspended, and the supernatant was used after centrifugation

23839

mAU

600

-1000 Minutes 14

Acidic variants Basic variants

Lysine truncationvariants

KK

K

Column: ProSwift WCX-1S, 4.6 × 50 mmEluents: A. 10 mM Sodium phosphate (pH 7.6) B. 1 M Sodium chloride in eluent A

Gradient: 5–10%Bin10min,100%Bfor4min

FlowRate: 1 mL/min

Inj. Volume: 10 µLTemperature: 30 °CDetection: UV, 214 nmSample: MAb 5 mg/mL

0 1 2 3 4 5 6 7 8 9 10 11 12-100

0

100

200

300

400

500

600

mAU

280

nm

Minutes

0.223

0.208

0.2010.2090.215

1

23

10 µg20 µg

80 µg120 µg

40 µg

Basic variants

00.5 1 1.5 2 2.5

0.15

0.25

Log Sample Load (µg)

PW1/

2 (m

in)

24349


Inj. Volume: Variable2–24µLTemperature: 30 °CDetection: UV, 280 nmSample: MAb 5 mg/mL

Column: ProSwift WCX-1S, 1 × 50 mmEluents: A. 10 mM Sodium phosphate, pH 7.6 B. 1 M NaCl in eluent AGradient: 1–15%Bin7.5minFlowRate: 0.2 mL/min


Figure 17. Comparison ProSwift WCX with competitor C column; separation of monoclonal antibody (MAb).

STABIlITy AND RUggeDNeSS

Figure 18. ProSwift RP-1S robustness.

CATION exChANge— COMPeTITOR COMPARISON

Figure 16. Comparison ProSwift WCX with competitor C column; separation of protein mixture.

24495

Minutes0 1 2 3 4 5 6 7 8

-100

900

mAU

ProSwift WCX

Competitor C

Column: ProSwift WCX, 4.6 × 50 mm FlowRate: 2 mL/minGradient: 1–100%Bin5min

Column: Competitor C, 5 × 50 mm FlowRate: 1 mL/minGradient: 1–50%Bin5min, 0.5 M NaCl max limit

Eluents: A. 10 mM Sodium phosphate, pH 7.6 B. 1 M NaCl in eluent AInj. Volume: 10 µL Detection: UV, 214 nm

Sample: Mixture of three proteins, 1mg/mL each 1. Ribonuclease A 2. Cytochrome C 3. Lysozyme

24496

Minutes

-100

600

mAU

-100

600

mAU

Columns: ProSwift WCX, 4.6 × 50 mm, Competitor C, 5 × 50 mm Eluents: A. 10 mM Sodium phosphate, pH 7.6 B. 1 M NaCl in eluent AFlowRate: 1 mL/min

Competitor C1 mL/min

ProSwift WCX1 mL/min

0 5 10 14

0 5

?

10 14

0 5 10 14-50

500

mAUProSwift WCX

2 mL/min



Inj. Volume: 10 µLDetection: UV, 214 nmSample: MAb 5 mg/mL

GradientFlowRate:1mL/min5–10%Bin10minGradientFlowRate:2mL/min:5–10%Bin5min

0

mAU

Minutes1 2 3 4

Injection 6

Injection 506

Injection 206

Injection 106

Injection 306

12

3 45

1 0.074 0.068 2 0.078 0.083 3 0.116 0.133 4 0.069 0.089 5 0.104 0.095

Peak # PWHH* Inj 6 PWHH Inj 506

* PWHH = Peak width at half height

Column: ProSwift RP-1S, 4.6 × 50 mm Flow Rate: 4 mL/minEluents: A. DI H2O/CH3CN, 95:5 (v/v) + 0.1% TFA B. DI H2O/CH3CN, 5:95 (v/v) + 0.1% TFA

Gradient: 1% B for 1 min, 1–75% B in 2.5 min

Inj. Volume: 10 µLDetection: UV, 214 nm

Peaks: 1. Ribonuclease A 1 mg/mL 2. Cytochrome C 0.5 3. BSA 1 4. Carbonic anhydrase 1 5. Ovalbumin 1

Sample was injected every 50 gradient cycles.

23845


Chromeleon and UltiMate are registered trademarks and ProSwift is a trademark of Dionex Corporation.

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Figure 19. Stability of WCX-1S.

Figure 20. Stability of SAX-1S.

CONClUSION• ProSwiftmonolithsareanewfamilyofcolumnsdevelopedforprotein

separations. They offer high speed, high resolution, and high loading capacity.

• ProSwiftmonolithcolumnsareavailablewithreversed-phaseandion-exchange chemistries.

• ProSwiftRPcolumnofferingsincludeRP-1S,RP-2H,andRP-3Ueach with different pore sizes.

• ProSwiftion-exchangecolumnsincludeWAX-1S,SAX-1Sand WCX-1S, available in 4.6 × 50 mm (all) and 1 × 50 mm (WAX-1S and WCX 1S) dimensions. ProSwift SCX (4.6 × 50 mm) and ProSwift SAX (1 mm)columns are in development.

•ProSwiftRPandIEXcolumnswerecomparedwithleadingbiocol-umns for various applications. The results confirmed that ProSwift columns performed better with a higher efficiency and resolution even at a higher flow rate, which results in higher productivity (RP comparisons:Figures6,7,8,andIEXcomparisons:Figures12,13,16, 17)

• Earlier,weintroduced1mmcolumndimensionsforWAXandWCXchemistries. Currently, we are developing SAX as well as RP chem-istries. These 1mm format columns offer improved sensitivity and reduce solvent consumption. Due to the high capacity of ProSwift IEX columns, they are ideally suited to be used in the first dimension in a multidimensional chromatography separation.

• ThelowbackpressuresinherenttoProSwiftcolumnsenabletheuseof high flow rates, resulting in high-throughput chromatographic separations.

•ProSwiftcolumnsofferexcellentstability,reproducibilityandoverallperformance.

0

mAU

Minutes1 2 3 4 5 6 7 8

7

90

170

251

337

421

13

Inj #

Column: ProSwift WCX-1S, 4.6 × 50 mmEluents: A. 10 mM Sodium phosphate, pH 7.6 B. 1 M NaCl in eluent A

Gradient: 0–100%Bin5min

FlowRate: 2 mL/min

2

24352

Inj. Volume: 10 µLDetection: UV, 214 nmSample: Protein mix, 1 mg/mL

Peaks: 1. Ribonuclease A 2. Cytochrome C 3. Lysozyme

0

mAU

Minutes3 6 9

Column: ProSwift SAX-1S, 4.6 × 50 mmEluents: A. 10 mM Tris, pH 7.6 B. 1 M NaCl in eluent A

Gradient: 0–50%Bin5min


4152302402502703803

1

2

3

4Inj #

24351

Inj. Volume: 10 µLTemperature: 30 °CDetection: UV, 214 nmSample: Protein mix 1 mg/mL

Peaks: 1. Myoglobin 2. Conalbumin 3.Ovalbumin 4. Trypsin Inhibitor

Documents

New ProSwift Monolith Reversed-Phase and Ion-Exchange